Controlled-release CNP agonists with low initial NPR-B activity

ABSTRACT

The present invention relates to a controlled-release CNP agonist from which CNP agonist is released with a release half-life of at least 6 hours under physiological conditions and which controlled-release CNP agonist has an EC50 that is at least 20-fold higher than the EC50 of the corresponding free CNP agonist and which released CNP agonist has an EC50 that is at most 3-fold higher than the EC50 of the corresponding free CNP agonist; to pharmaceutical compositions comprising said controlled-release CNP agonist; their use; and to methods of treatment.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a US national stage of PCT/EP2017/050217filed Jan. 5, 2017, incorporated by reference in its entirety for allpurposes, which claims the benefit of EP 16150631.6 filed Jan. 8, 2016;EP 16179291.6 filed Jul. 13, 2016; and EP 16191462.7 filed Sep. 29,2016.

REFERENCE TO SEQUENCE LISTING

This application includes an electronic sequence listing in a file named“514992_SEQ.TXT”, created on Jun. 21, 2018 and containing 48,226 bytes,which is incorporated by reference.

The present invention relates to a controlled-release CNP agonist fromwhich CNP agonist is released with a release half-life of at least 6hours under physiological conditions and which controlled-release CNPagonist has an EC₅₀ that is at least 20-fold higher than the EC₅₀ of thecorresponding free CNP agonist and which released CNP agonist has anEC₅₀ that is at most 3-fold higher than the EC₅₀ of the correspondingfree CNP agonist; to pharmaceutical compositions comprising saidcontrolled-release CNP agonist; their use; and to methods of treatment.

Achondroplasia (ACH) is caused by a gain-of-function mutation in FGFR3.Binding of CNP to its receptor, natriuretic-peptide receptor B (NPR-B),inhibits FGFR3 downstream signaling and thus triggers endochondralgrowth and skeletal overgrowth, as observed in both mice and humansoverexpressing CNP. Overproduction of CNP in the cartilage or continuousdelivery of CNP through intravenous (iv) infusion normalizes thedwarfism of achondroplasic mice, suggesting that administration of CNPat supraphysiological levels is a strategy for treating ACH.

However, given its short half-life of CNP-22 (2 min after ivadministration) CNP as a therapeutic agent is challenging in a pediatricpopulation because it would require continuous infusion. Furthermore, asCNP is extensively inactivated in the subcutaneous tissue iv infusion isrequired.

Increasing exposure to efficacious levels of the natriuretic peptide CNPis challenging. As natriuretic peptides are a family of hormones thatmay affect blood volume and blood pressure, an increase in dose may beassociated with cardiovascular adverse effects. A study in healthyvolunteers demonstrated that CNP injection caused a transient butsignificant decrease in both systolic and diastolic blood pressure witha significant increase in heart rate (Igaki, et al. Hypertens Res 1998;21: 7-13). Similarly, a CNP variant with increased NEP resistance(BMN-111) in development for the treatment of achondroplasia, hasdemonstrated mild hypotension in a Phase 1 study in healthy volunteers(BioMarin press release Sep. 26, 2012). Studies of BMN-111 in animalsand man have demonstrated that as the dose increases, arterial bloodpressure (BP) drops and heart rate (HR) increases.

CNP produces hemodynamic effects in mice, nonhuman primates, rats, dogs,and humans. In order to evaluate the cardiovascular effects of variousCNP variants, anesthetized wild-type FVB/nJ male mice were fitted with apressure monitoring catheter connected to a telemetry transmitter. Allvariants showed similar BP-reducing and HR-increasing activity. In mostanimals, effects were observed within 5 minutes of subcutaneousadministration, with maximal drop in MAP occurring between 5 and 20minutes postdose. This timing correlated well with the maximumconcentration of the CNP variants, and demonstrated a clear PK/PDrelationship for this physiologic response. Because the hemodynamicresponses were similar between the doses and variants tested,cardiovascular activity was determined not to be a differentiatingproperty (Wendt et. al. J Pharmacol Exp Ther 353:132-149, April 2015).

In addition to investigating various variants of CNP, different CNPconjugates were obtained by conjugating the CNP moiety to either PEG orproteinaceous compounds. These PEGylated and chimeric CNP exhibited asimilar hemodynamic response as observed for the non-PEGylated CNPvariants (Wendt, J Pharmacol Exp Ther 353:132-149, April 2015) Thereforeincreasing the dose of a drug having CNP activity to increase drugexposure may be associated with unacceptable cardiovascular sideeffects, such as hypotension.

In summary, there is a need for a more efficacious and safer CNPtreatment.

It is therefore an object of the present invention to at least partiallyovercome the shortcomings described above.

This object is achieved with a controlled-release CNP agonist from whichCNP agonist is released with a release half-life of at least 6 hoursunder physiological conditions and which controlled-release CNP agonisthas an EC₅₀ that is at least 20-fold higher than the EC₅₀ of thecorresponding free CNP agonist and which released CNP agonist has anEC₅₀ that is at most 3-fold higher than the EC₅₀ of the correspondingfree CNP agonist.

It was surprisingly found that such reversible inactivation of CNP'saffinity towards NPR-B allows the administration of a higher, moreefficacious dose with reduced cardiovascular side effects, such ashypotension.

It was furthermore surprisingly found that a continuous release of CNPis more efficacious than a once-daily bolus injection, so the continuousrelease from the controlled-release CNP agonist even further increasesefficacy.

Within the present invention the terms are used having the meaning asfollows.

As used herein the term “CNP agonist” refers to any compound thatactivates natriuretic peptide receptor B (NPR-B) and has an EC₅₀ that isat most 50-fold higher than the NPR-B activity of CNP-22 (SEQ ID NO:1).

As used herein “EC₅₀” with regard to controlled-release CNP agonist andCNP agonist refers to the concentration of controlled-release CNPagonist and CNP agonist with which a half-maximum cGMP production iselicited. NPR-B activity in the form of its EC₅₀ of thecontrolled-release CNP agonist, of the released CNP agonist and ofCNP-22 is measured by cultivating NIH-3T3 (Murine Embryo Fibroblast cellline) cells which express NPR-B on their cell surface, incubating thecells with the controlled-release CNP agonist, the correspondingreleased CNP agonist or CNP-22, respectively, and determining theintracellular production of the second messenger cGMP with a standardcGMP assay. In particular the assay is performed as follows:

-   -   (1) murine NIH-3T3 cells expressing endogenous NPR-B are        cultivated in DMEM F-12 medium with 5% FBS and 5 mM glutamine at        37° C. and 5% CO₂;    -   (2) for each assay 50,000 cells are resuspended in Dulbecco's        PBS with IBMX and incubated with either the controlled-release        CNP agonist, the corresponding released CNP agonist or CNP-22;        each in different concentrations;    -   (3) after incubating for 30 min at 37° C. and 5% CO₂, the cells        are lysed and cGMP levels are determined; and    -   (4) generating an EC₅₀ value from the determined cGMP levels.

Preferably, the IBMX concentration in step (2) is 0.5 mM.

Step (3) can be performed using any assay for measuring cGMP which is astandard procedure well known to the person skilled in the art.Preferably step (3) is done with a cGMP TR-FRET assay, more preferablywith the cGMP TR-FRET assay from Cisbio, Cat. No. 62GM2PEB.

As during such experiments the controlled-release CNP agonist releases acertain amount of CNP agonist which released CNP agonist would distortthe results, measurements for the NPR-B activity of thecontrolled-release CNP agonist are preferably made in the form of astable analog which does not release CNP agonist.

As used herein the term “controlled-release CNP agonist” refers to anycompound, conjugate, crystal or admixture that comprises at least oneCNP agonist and from which the at least one CNP agonist is released witha release half-life of at least 6 hours.

As used herein the term “CNP agonist equivalent” refers to molar contentof CNP agonist comprised in a controlled-release CNP agonist.

As used herein the term “release half-life” refers to the time neededuntil half of all CNP agonist molecules are released from thecontrolled-release CNP agonist under physiological conditions.

As used herein the term “CNP” refers to all CNP polypeptides, preferablyfrom mammalian species, more preferably from human and mammalianspecies, more preferably from human and murine species, as well as theirvariants, analogs, orthologs, homologs, and derivatives and fragmentsthereof, that are characterized by regulating the growth, proliferationand differentiation of cartilaginous growth plate chondrocytes.Preferably, the term “CNP” refers to the CNP polypeptide of SEQ ID NO:24as well as its variants, homologs and derivatives exhibiting essentiallythe same biological activity, i.e. regulating the growth, proliferationand differentiation of cartilaginous growth plate chondrocytes. Morepreferably, the term “CNP” refers to the polypeptide of SEQ ID NO:24. Inanother preferred embodiment the term “CNP” refers to the polypeptide ofSEQ ID NO:20. In another preferred embodiment the term “CNP” refers tothe polypeptide of SEQ ID NO:21. In another preferred embodiment theterm “CNP” refers to the polypeptide of SEQ ID NO:22. In anotherpreferred embodiment the term “CNP” refers to the polypeptide of SEQ IDNO:23. In another preferred embodiment the term “CNP” refers to thepolypeptide of SEQ ID NO:30.

Naturally occurring CNP-22 (SEQ ID NO: 1) has the following sequence:

GLSKGCFGLKLDRIGSMSGLGC,

wherein the cysteines at position 6 and 22 are connected through adisulfide-bridge, as illustrated in FIG. 1.

SEQ ID NO:24 has the following sequence:

LQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC,

wherein the cysteines at position 22 and 38 are connected through adisulfide-bride.

The term “CNP” also includes all CNP variants, analogs, orthologs,homologs and derivatives and fragments thereof as disclosed in WO2009/067639 A2 and WO 2010/135541 A2, which are herewith incorporated byreference.

Accordingly, the term “CNP” also refers preferably to the followingpeptide sequences:

SEQ ID NO: 2 (CNP-53):DLRVDTKSRAAWARLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSG LGC;SEQ ID NO: 3 (G-CNP-53):GDLRVDTKSRAAWARLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMS GLGC;SEQ ID NO: 4 (M-CNP-53):MDLRVDTKSRAAWARLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMS GLGC;SEQ ID NO: 5 (P-CNP-53):PDLRVDTKSRAAWARLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMS GLGC;SEQ ID NO: 6 (CNP-53 M48N):DLRVDTKSRAAWARLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSNSG LGC;SEQ ID NO: 7 (CNP-53 Δ15-31): DLRVDTKSRAAWARGLSKGCFGLKLDRIGSMSGLGC;SEQ ID NO: 8 (CNP-52):LRVDTKSRAAWARLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGL GC;SEQ ID NO: 9 (CNP-51):RVDTKSRAAWARLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLG C;SEQ ID NO: 10 (CNP-50):VDTKSRAAWARLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLG C;SEQ ID NO: 11 (CNP-49):DTKSRAAWARLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC;SEQ ID NO: 12 (CNP-48):TKSRAAWARLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC;SEQ ID NO: 13 (CNP-47): KSRAAWARLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC;SEQ ID NO: 14 (CNP-46): SRAAWARLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC;SEQ ID NO: 15 (CNP-45): RAAWARLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC;SEQ ID NO: 16 (CNP-44): AAWARLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC;SEQ ID NO: 17 (CNP-44 Δ14-22): AAWARLLQEHPNAGLSKGCFGLKLDRIGSMSGLGC;SEQ ID NO: 18 (CNP-44 Δ15-22): AAWARLLQEHPNARGLSKGCFGLKLDRIGSMSGLGC;SEQ ID NO: 19 (CNP-43): AWARLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC;SEQ ID NO: 20 (CNP-42): WARLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC;SEQ ID NO: 21 (CNP-41): ARLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC;SEQ ID NO: 22 (CNP-40): RLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC;SEQ ID NO: 23 (CNP-39): LLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC;SEQ ID NO: 24 (CNP-38): LQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC;SEQ ID NO: 25 (CNP-37): QEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC;SEQ ID NO: 26 (CNP-37 Q1pQ, wherein pQ = pyroglutamate):pQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC; SEQ ID NO: 27 (G-CNP-37):GQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC; SEQ ID NO: 28 (P-CNP-37):PQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC; SEQ ID NO: 29 (M-CNP-37):MQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC; SEQ ID NO: 30 (PG-CNP-37):PGQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC; SEQ ID NO: 31 (MG-CNP-37):MGQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC; SEQ ID NO: 32 (CNP-37 M32N):QEHPNARKYKGANKKGLSKGCFGLKLDRIGSNSGLGC; SEQ ID NO: 33 (G-CNP-37 M32N):GQEHPNARKYKGANKKGLSKGCFGLKLDRIGSNSGLGC; SEQ ID NO: 34 (G-CNP-37 K14Q):GQEHPNARKYKGANQKGLSKGCFGLKLDRIGSMSGLGC; SEQ ID NO: 35 (G-CNP-37 K14P):GQEHPNARKYKGANPKGLSKGCFGLKLDRIGSMSGLGC;SEQ ID NO: 36 (G-CNP-37 K14Q, Δ15):GQEHPNARKYKGANQGLSKGCFGLKLDRIGSMSGLGC;SEQ ID NO: 37 (G-CNP-37 K14Q, K15Q):GQEHPNARKYKGANQQGLSKGCFGLKLDRIGSMSGLGC; SEQ ID NO: 38 (CNP-36):EHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC; SEQ ID NO: 39 (CNP-35):HPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC; SEQ ID NO: 40 (CNP-34):PNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC; SEQ ID NO: 41 (CNP-33):NARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC; SEQ ID NO: 42 (CNP-32):ARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC; SEQ ID NO: 43 (CNP-31):RKYKGANKKGLSKGCFGLKLDRIGSMSGLGC; SEQ ID NO: 44 (CNP-30):KYKGANKKGLSKGCFGLKLDRIGSMSGLGC; SEQ ID NO: 45 (CNP-29):YKGANKKGLSKGCFGLKLDRIGSMSGLGC; SEQ ID NO: 46 (CNP-28):KGANKKGLSKGCFGLKLDRIGSMSGLGC; SEQ ID NO: 47 (GHKSEVAHRF-CNP-28):GHKSEVAHRFKGANKKGLSKGCFGLKLDRIGSMSGLGC; SEQ ID NO: 48 (CNP-27):GANKKGLSKGCFGLKLDRIGSMSGLGC; SEQ ID NO: 49 (CNP-27 K4Q, K5Q):GANQQGLSKGCFGLKLDRIGSMSGLGC; SEQ ID NO: 50 (CNP-27 K4R, K5R):GANRRGLSKGCFGLKLDRIGSMSGLGC; SEQ ID NO: 51 (CNP-27 K4P, K5R):GANPRGLSKGCFGLKLDRIGSMSGLGC; SEQ ID NO: 52 (CNP-27 K4S, K5S):GANSSGLSKGCFGLKLDRIGSMSGLGC; SEQ ID NO: 53 (CNP-27 K4P, K5R):GANGANPRGLSRGCFGLKLDRIGSMSGLGC; SEQ ID NO: 54 (CNP-27 K4R, K5R, K9R):GANRRGLSRGCFGLKLDRIGSMSGLGC; SEQ ID NO: 55 (CNP-27 K4R, K5R, K9R, M22N):GANRRGLSRGCFGLKLDRIGSNSGLGC; SEQ ID NO: 56 (P-CNP-27 K4R, K5R, K9R):PGANRRGLSRGCFGLKLDRIGSMSGLGC; SEQ ID NO: 57 (M-CNP-27 K4R, K5R, K9R):MGANRRGLSRGCFGLKLDRIGSMSGLGC; SEQ ID NO: 58 (HSA fragment-CNP-27):GHKSEVAHRFKGANKKGLSKGCFGLKLDRIGSMSGLG;SEQ ID NO: 59 (HSA fragment-CNP-27 M22N):GHKSEVAHRFKGANKKGLSKGCFGLKLDRIGSNSGLGC;SEQ ID NO: 60 (M-HSA fragment-CNP-27):MGHKSEVAHRFKGANKKGLSKGCFGLKLDRIGSMSGLGC;SEQ ID NO: 61 (P-HSA fragment-CNP-27):PGHKSEVAHRFKGANKKGLSKGCFGLKLDRIGSMSGLGC; SEQ ID NO: 62 (CNP-26):ANKKGLSKGCFGLKLDRIGSMSGLGC; SEQ ID NO: 63 (CNP-25):NKKGLSKGCFGLKLDRIGSMSGLGC; SEQ ID NO: 64 (CNP-24):KKGLSKGCFGLKLDRIGSMSGLGC; SEQ ID NO: 65 (CNP-23):KGLSKGCFGLKLDRIGSMSGLGC; SEQ ID NO: 66 (R-CNP-22):RGLSKGCFGLKLDRIGSMSGLGC; SEQ ID NO: 67 (ER-CNP-22):ERGLSKGCFGLKLDRIGSMSGLGC; SEQ ID NO: 68 (R-CNP-22 K4R):RGLSRGCFGLKLDRIGSMSGLGC; SEQ ID NO: 69 (ER-CNP-22 4KR):ERGLSRGCFGLKLDRIGSMSGLGC; SEQ ID NO: 70 (RR-CNP-22):RRGLSRGCFGLKLDRIGSMSGLGC; SEQ ID NO: 71 (HRGP fragment-CNP-22):GHHSHEQHPHGANQQGLSKGCFGLKLDRIGSMSGLGC;SEQ ID NO: 72 (HRGP fragment-CNP-22):GAHHPHEHDTHGANQQGLSKGCFGLKLDRIGSMSGLGC;SEQ ID NO: 73 (HRGP fragment-CNP-22):GHHSHEQHPHGANPRGLSKGCFGLKLDRIGSMSGLGC;SEQ ID NO: 74 (IgG₁(F_(c)) fragment-CNP-22):GQPREPQVYTLPPSGLSKGCFGLKLDRIGSMSGLGC;SEQ ID NO: 75 (HSA fragment-CNP-22):GQHKDDNPNLPRGANPRGLSKGCFGLKLDRIGSMSGLGC;SEQ ID NO: 76 (HSA fragment-CNP-22):GERAFKAWAVARLSQGLSKGCFGLKLDRIGSMSGLGC;SEQ ID NO: 77 (osteocrin NPR C inhibitor  fragment-CNP22):FGIPMDRIGRNPRGLSKGCFGLKLDRIGSMSGLGC;SEQ ID NO: 78 (FGF2 heparin-binding domain  fragment-CNP22):GKRTGQYKLGSKTGPGPKGLSKGCFGLKLDRIGSMSGLGC;SEQ ID NO: 79 (IgG₁(F_(c)) fragment-CNP-22 K4R):GQPREPQVYTGANQQGLSRGCFGLKLDRIGSMSGLGC;SEQ ID NO: 80 (HSA fragment-CNP-22 K4R):GVPQVSTSTGANQQGLSRGCFGLKLDRIGSMSGLGC;SEQ ID NO: 81 (fibronectin fragment-CNP-22 K4R):GQPSSSSQSTGANQQGLSRGCFGLKLDRIGSMSGLGC;SEQ ID NO: 82 (fibronectin fragment-CNP-22 K4R):GQTHSSGTQSGANQQGLSRGCFGLKLDRIGSMSGLGC;SEQ ID NO: 83 (fibronectin fragment-CNP-22 K4R):GSTGQWHSESGANQQGLSRGCFGLKLDRIGSMSGLGC;SEQ ID NO: 84 (zinc finger fragment-CNP-22 K4R):GSSSSSSSSSGANQQGLSRGCFGLKLDRIGSMSGLGC; SEQ ID NO: 85 (CNP-21):LSKGCFGLKLDRIGSMSGLGC; SEQ ID NO: 86 (CNP-20): SKGCFGLKLDRIGSMSGLGC;SEQ ID NO: 87 (CNP-19): KGCFGLKLDRIGSMSGLGC; SEQ ID NO: 88 (CNP-18):GCFGLKLDRIGSMSGLGC; SEQ ID NO: 89 (CNP-17): CFGLKLDRIGSMSGLGC;SEQ ID NO: 90 (BNP fragment-CNP-17-BNP fragment):SPKMVQGSGCFGLKLDRIGSMSGLGCKVLRRH; SEQ ID NO: 91 (CNP-38 L1G):GQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC;SEQ ID NO: 92 (Ac-CNP-37; wherein Ac = acetyl):Ac-QEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC.

It is understood that the equivalents of the cysteines in positions 22and 38 of SEQ ID NO:24 are also connected through a disulfide-bridge inSEQ ID NOs: 2 to 92.

More preferably, the term “CNP” refers to the sequence of SEQ ID:NOs 2,19, 20, 21, 22, 23, 24, 25, 26, 30, 32, 38, 39, 40, 41, 42, 43, 91, 92.Even more preferably, the term “CNP” refers to the sequence of SEQID:NOs 23, 24, 25, 26, 38, 39, 91 and 92. In a particularly preferredembodiment the term “CNP” refers to the sequence of SEQ ID NO:24.

In another preferred embodiment the term “CNP” refers to a sequence ofSEQ ID NO:93

QEHPNARX₁YX₂GANX₃X₄GLSX₅GCFGLX₆LDRIGSMSGLGC,

wherein X₁, X₂, X₃, X₄, X₅ and X₆ are independently of each otherselected from the group consisting of K, R, P, S and Q, with theprovision that at least one of X₁, X₂, X₃, X₄, X₅ and X₆ is selectedfrom the group consisting of R, P, S and Q; preferably X₁, X₂, X₃, X₄,X₅ and X₆ are selected from the group consisting of K and R, with theprovision that at least one of X₁, X₂, X₃, X₄, X₅ and X₆ is R;

even more preferably to a sequence of SEQ ID NO:94

QEHPNARKYKGANX₁X₂GLSX₃GCFGLX₄LDRIGSMSGLGC,

wherein X₁, X₂, X₃ and X₄ are independently of each other selected fromthe group consisting of K, R, P, S and Q, with the provision that atleast one of X₁, X₂, X₃ and X₄ is selected from the group consisting ofR, P, S and Q; preferably X₁, X₂, X₃ and X₄ are selected from K and R,with the provision that at least one of X₁, X₂, X₃ and X₄ is R;

and most preferably to a sequence of SEQ ID NO:95

QEHPNARKYKGANX₁X₂GLSKGCFGLKLDRIGSMSGLGC,

wherein X₁X₂ are selected from the group consisting of KR, RK, KP, PK,SS, RS, SR, QK, QR, KQ, RQ, RR and QQ.

It is understood that in all CNP sequences given in this specificationthe equivalents of the cysteines in positions 22 and 38 of SEQ ID NO:24are also connected through a disulfide-bridge in SEQ ID NOs: 93 to 95.

It is understood that the present invention also encompasses CNPvariants in which any one or more, up to all, residues susceptible todeamidation or a deamidation-like reaction (e.g., isomerization) may beconverted to other residue(s) via deamidation or a deamidation-likereaction to any extent, up to 100% conversion per converted residue. Incertain embodiments, the disclosure encompasses CNP variants in which:

(1) any one or more, up to all, asparagine (Asn/N) residues may beconverted to aspartic acid or aspartate, and/or to isoaspartic acid orisoaspartate, via deamidation up to about 5%, 10%, 20%, 30%, 40%, 50%,60%, 70%, 80%, 90% or 100% conversion per converted residue; or

(2) any one or more, up to all, glutamine (Gln/Q) residues may beconverted to glutamic acid or glutamate, and/or to isoglutamic acid orisoglutamate, via deamidation up to about 5%, 10%, 20%, 30%, 40%, 50%,60%, 70%, 80%, 90% or 100% conversion per converted residue; or

(3) any one or more, up to all, aspartic acid or aspartate (Asp/D)residues may be converted to isoaspartic acid or isoaspartate via adeamidation-like reaction (also called isomerization) up to about 5%,10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% conversion perconverted residue; or

(4) any one or more, up to all, glutamic acid or glutamate (Glu/E)residues may be converted to isoglutamic acid or isoglutamate via adeamidation-like reaction (also called isomerization) up to about 5%,10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% conversion perconverted residue; or

(5) the N-terminal glutamine (if present) may be converted intopyroglutamate up to about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%,90% or 100% conversion; or

(6) a combination of the above.

As used herein, the term “CNP polypeptide variant” refers to apolypeptide from the same species that differs from a reference CNPpolypeptide. Preferably, such reference CNP polypeptide sequence is thesequence of SEQ ID NO:24. Generally, differences are limited so that theamino acid sequence of the reference and the variant are closely similaroverall and, in many regions, identical. Preferably, CNP polypeptidevariants are at least 70%, 80%, 90%, or 95% identical to a reference CNPpolypeptide, preferably the CNP polypeptide of SEQ ID NO:24. By apolypeptide having an amino acid sequence at least, for example, 95%“identical” to a query amino acid sequence, it is intended that theamino acid sequence of the subject polypeptide is identical to the querysequence except that the subject polypeptide sequence may include up tofive amino acid alterations per each 100 amino acids of the query aminoacid sequence. These alterations of the reference sequence may occur atthe amino (N-terminal) or carboxy terminal (C-terminal) positions of thereference amino acid sequence or anywhere between those terminalpositions, interspersed either individually among residues in thereference sequence or in one or more contiguous groups within thereference sequence. The query sequence may be an entire amino acidsequence of the reference sequence or any fragment specified asdescribed herein. Preferably, the query sequence is the sequence of SEQID NO:24.

Such CNP polypeptide variants may be naturally occurring variants, suchas naturally occurring allelic variants encoded by one of severalalternate forms of a CNP occupying a given locus on a chromosome or anorganism, or isoforms encoded by naturally occurring splice variantsoriginating from a single primary transcript. Alternatively, a CNPpolypeptide variant may be a variant that is not known to occurnaturally and that can be made by mutagenesis techniques known in theart.

It is known in the art that one or more amino acids may be deleted fromthe N-terminus or C-terminus of a bioactive peptide or protein withoutsubstantial loss of biological function. Such N- and/or C-terminaldeletions are also encompassed by the term CNP polypeptide variant. Itis also recognized by one of ordinary skill in the art that some aminoacid sequences of CNP polypeptides can be varied without significanteffect of the structure or function of the peptide. Such mutants includedeletions, insertions, inversions, repeats, and substitutions selectedaccording to general rules known in the art so as to have little effecton activity. For example, guidance concerning how to make phenotypicallysilent amino acid substitutions is provided in Bowie et al. (1990),Science 247:1306-1310, which is hereby incorporated by reference in itsentirety, wherein the authors indicate that there are two mainapproaches for studying the tolerance of the amino acid sequence tochange.

The term CNP polypeptide also encompasses all CNP polypeptides encodedby CNP analogs, orthologs, and/or species homologs. As used herein, theterm “CNP analog” refers to CNP of different and unrelated organismswhich perform the same functions in each organism, but which did notoriginate from an ancestral structure that the organisms' ancestors hadin common. Instead, analogous CNPs arose separately and then laterevolved to perform the same or similar functions. In other words,analogous CNP polypeptides are polypeptides with quite different aminoacid sequences but that perform the same biological activity, namelyregulating the growth, proliferation and differentiation ofcartilaginous growth plate chondrocytes.

As used herein the term “CNP ortholog” refers to CNP within twodifferent species which sequences are related to each other via a commonhomologous CNP in an ancestral species, but which have evolved to becomedifferent from each other.

As used herein, the term “CNP homolog” refers to CNP of differentorganisms which perform the same functions in each organism and whichoriginate from an ancestral structure that the organisms' ancestors hadin common. In other words, homologous CNP polypeptides are polypeptideswith quite similar amino acid sequences that perform the same biologicalactivity, namely regulating the growth, proliferation anddifferentiation of cartilaginous growth plate chondrocytes. Preferably,CNP polypeptide homologs may be defined as polypeptides exhibiting atleast 40%, 50%, 60%, 70%, 80%, 90% or 95% identity to a reference CNPpolypeptide, preferably the CNP polypeptide of SEQ ID NO:24.

Thus, a CNP polypeptide according to the invention may be, for example:(i) one in which at least one of the amino acid residues is substitutedwith a conserved or non-conserved amino acid residue, preferably aconserved amino acid residue, and such substituted amino acid residuemay or may not be one encoded by the genetic code; and/or (ii) one inwhich at least one of the amino acid residues includes a substituentgroup; and/or (iii) one in which the CNP polypeptide is fused withanother compound, such as a compound to increase the half-life of thepolypeptide (for example, polyethylene glycol); and/or (iv) one in whichadditional amino acids are fused to the CNP polypeptide, such as an IgGFc fusion region peptide or leader or secretory sequence or a sequencewhich is employed for purification of the above form of the polypeptideor a pre-protein sequence.

As used herein, the term “CNP polypeptide fragment” refers to anypeptide comprising a contiguous span of a part of the amino acidsequence of a CNP polypeptide, preferably the polypeptide of SEQ IDNO:24.

More specifically, a CNP polypeptide fragment comprises at least 6, suchas at least 8, at least 10 or at least 17 consecutive amino acids of aCNP polypeptide, more preferably of the polypeptide of SEQ ID NO:24. ACNP polypeptide fragment may additionally be described as sub-genuses ofCNP polypeptides comprising at least 6 amino acids, wherein “at least 6”is defined as any integer between 6 and the integer representing theC-terminal amino acid of a CNP polypeptide, preferably of thepolypeptide of SEQ ID No:24. Further included are species of CNPpolypeptide fragments at least 6 amino acids in length, as describedabove, that are further specified in terms of their N-terminal andC-terminal positions. Also encompassed by the term “CNP polypeptidefragment” as individual species are all CNP polypeptide fragments, atleast 6 amino acids in length, as described above, that may beparticularly specified by a N-terminal and C-terminal position. That is,every combination of a N-terminal and C-terminal position that afragment at least 6 contiguous amino acid residues in length couldoccupy, on any given amino acid sequence of a CNP polypeptide,preferably the CNP polypeptide of SEQ ID:NO24 is included in the presentinvention.

The term “CNP” also includes poly(amino acid) conjugates which have asequence as described above, but having a backbone that comprises bothamide and non-amide linkages, such as ester linkages, like for exampledepsipeptides. Depsipeptides are chains of amino acid residues in whichthe backbone comprises both amide (peptide) and ester bonds.Accordingly, the term “side chain” as used herein refers either to themoiety attached to the alpha-carbon of an amino acid moiety, if theamino acid moiety is connected through amine bonds such as inpolypeptides, or to any carbon atom-comprising moiety attached to thebackbone of a poly(amino acid) conjugate, such as for example in thecase of depsipeptides. Preferably, the term “CNP” refers to polypeptideshaving a backbone formed through amide (peptide) bonds.

As the term CNP includes the above-described variants, analogs,orthologs, homologs, derivatives and fragments of CNP, all references tospecific positions within a reference sequence also include theequivalent positions in the variants, analogs, orthologs, homologs,derivatives and fragments of a CNP moiety, even if not specificallymentioned.

As used herein, the term “ring moiety” refers to the stretch ofconsecutive amino acid residues of the CNP drug or moiety that islocated between two cysteine residues that form an intramoleculardisulfide bridge or between homologous amino acid residues which areconnected through a chemical linker. Preferably, the ring moiety islocated between two cysteine residues that form an intramoleculardisulfide bridge. These two cysteines correspond to the cysteines atposition 22 and position 38 in the sequence of CNP-38 (SEQ ID NO:24).Accordingly, amino acids 23 to 37 are located in said ring moiety, ifthe CNP drug or moiety has the sequence of CNP-38.

Independently of the length of the CNP moiety, the sequence of the ringmoiety of wild-type CNP is FGLKLDRIGSMSGLG (SEQ ID NO:96).

As described above, the term “CNP” relates to CNP drugs or moietieshaving different numbers of amino acids. The person skilled in the artunderstands that in CNP drugs or moieties of different lengths thepositions of equivalent amino acids vary and the skilled artisan willhave no difficulty identifying the two cysteines forming the disulfidebridge or their two homologous amino acid residues connected to eachother through a chemical linker in longer, shorter and/or otherwisemodified CNP versions.

As the term CNP includes the above-described variants, analogs,orthologs, homologs, derivatives and fragments of CNP, the term “ringmoiety” also includes the corresponding variants, analogs, orthologs,homologs, derivatives and fragments of the sequence of SEQ ID NO:96.Accordingly, all references to specific positions within a referencesequence also include the equivalent positions in variants, analogs,orthologs, homologs, derivatives and fragments of a CNP moiety, even ifnot explicitly mentioned.

As used herein, the term “random coil” refers to a peptide or proteinadopting/having/forming, preferably having, a conformation whichsubstantially lacks a defined secondary and tertiary structure asdetermined by circular dichroism spectroscopy performed in aqueousbuffer at ambient temperature, and pH 7.4. Preferably, ambienttemperature is about 20° C., i.e. between 18° C. and 22° C., mostpreferably ambient temperature is 20° C.

As used herein the term “micelle” means an aggregate of amphiphilicmolecules dispersed in a liquid colloid. In aqueous solution a typicalmicelle forms an aggregate with the hydrophilic moiety of the surfactantmolecules facing the surrounding solvent and the hydrophobic moiety ofthe surfactant molecule facing inwards, also called “normal-phasemicelle”. “Invers micelles” have the hydrophilic moiety facing inwardsand the hydrophobic moiety facing the surrounding solvent.

As used herein the term “liposome” refers to a vesicle, preferably aspherical vesicle, having at least one lipid bilayer. Preferably,liposomes comprise phospholipids, even more preferablyphosphatidylcholine. The term “liposome” refers to various structuresand sizes, such as, for example, to multilamellar liposome vesicles(MLV) having more than one concentric lipid bilayer with an averagediameter of 100 to 1000 nm, small unilamellar liposome vesicles (SUV)having one lipid bilayer and an average diameter of 25 to 100 nm, largeunilamellar liposome vesicles (LUV) having one lipid bilayer and anaverage diameter of about 1000 μm and giant unilamellar vesicles (GUV)having one lipid bilayer and an average diameter of 1 to 100 μm. Theterm “liposome” also includes elastic vesicles such as transferosomesand ethosomes, for example.

As used herein the term “aquasome” refers to spherical nanoparticleshaving a diameter of 60 to 300 nm that comprise at least three layers ofself-assembled structure, namely a solid phase nanocrystalline corecoated with an oligomeric film to which drug molecules are adsorbed withor without modification of the drug.

As used herein the term “ethosome” refers to lipid vesicles comprisingphospholipids and ethanol and/or isopropanol in relatively highconcentration and water, having a size ranging from tens of nanometersto micrometers.

As used herein the term “LeciPlex” refers to positively chargedphospholipid-based vesicular system which comprises soy PC, a cationicagent, and a bio-compatible solvent like PEG 300, PEG 400, diethyleneglycol monoethyl ether, tetrahydrofurfuryl alcohol polyethylene glycolether or 2-pyrrolidoneor N-methyl-2-pyrrolidone.

As used herein the term “niosome” refers to unilamellar or multilamellarvesicles comprising non-ionic surfactants.

As used herein the term “pharmacosome” refers to ultrafine vesicular,micellar or hexagonal aggregates from lipids covalently bound tobiologically active moieties.

As used herein the term “proniosome” refers to dry formulations ofsurfactant-coated carrier which on rehydration and mild agitation givesniosomes.

As used herein the term “polymersome” refers to an artificial sphericalvesicle comprising a membrane formed from amphiphilic synthetic blockcopolymers and may optionally comprise an aqueous solution in its core.A polymersome has a diameter ranging from 50 nm to 5 μm and larger. Theterm also includes syntosomes, which are polymersomes engineered tocomprise channels that allow certain chemicals to pass through themembrane into or out of the vesicle.

As used herein the term “sphingosome” refers to a concentric, bilayeredvesicle in which an aqueous volume is entirely enclosed by a membranouslipid bilayer mainly composed of natural or synthetic sphingolipid.

As used herein the term “transferosome” refers to ultraflexible lipidvesicles comprising an aqueous core that are formed from a mixture ofcommon polar and suitable edge-activated lipids which facilitate theformation of highly curved bilayers which render the transferosomehighly deformable.

As used herein the term “ufasome” refers to a vesicle comprisingunsaturated fatty acids.

As used herein the term “aptamer” refers to an oligonucleotide orpeptide molecule that binds a specific molecule. The term “aptamer”includes DNA, RNA, XNA and peptide aptamers.

As used herein, the term “oligonucleotide” refers to a short nucleicacid polymer of up to 100 bases.

As used herein the term “polypeptide” refers to a peptide comprising upto and including 50 amino acid monomers. Only for CNP drugs and CNPmoieties also sequences having more than 50 amino acids will be referredto as “polypeptide” for simplification.

As used herein the term “protein” refers to a peptide of more than 50amino acid residues.

Preferably a protein comprises at most 20000 amino acid residues, suchas at most 15000 amino acid residues, such as at most 10000 amino acidresidues, such as at most 5000 amino acid residues, such as at most 4000amino acid residues, such as at most 3000 amino acid residues, such asat most 2000 amino acid residues, such as at most 1000 amino acidresidues.

As used herein the terms “small molecule drug” and “small moleculebiologically active moiety” refer to drugs and biologically activemoieties that are organic compounds having a molecular weight of no morethan 1 kDa, such as up to 900 kDa.

As used herein the term “natural product” refers to purified organiccompounds isolated from natural sources that are produced by thepathways of primary or secondary metabolism.

As used herein the term “physiological conditions” refers to an aqueousbuffer at pH 7.4, 37° C.

As used herein the term “pharmaceutical composition” refers to acomposition containing one or more active ingredients, such as forexample the controlled-release CNP agonists of the present invention,and one or more excipients, as well as any product which results,directly or indirectly, from combination, complexation or aggregation ofany two or more of the ingredients of the composition, or fromdissociation of one or more of the ingredients, or from other types ofreactions or interactions of one or more of the ingredients.Accordingly, the pharmaceutical compositions of the present inventionencompass any composition made by admixing one or morecontrolled-release CNP agonists of the present invention and apharmaceutically acceptable excipient.

As used herein the term “liquid composition” refers to a mixturecomprising water-soluble controlled-release CNP agonist and one or moresolvents, such as water.

The term “suspension composition” relates to a mixture comprisingwater-insoluble controlled-release CNP agonist and one or more solvents,such as water.

As used herein, the term “dry composition” means that a pharmaceuticalcomposition is provided in a dry form. Suitable methods for drying arespray-drying and lyophilization, i.e. freeze-drying. Such drycomposition of controlled-release CNP agonist of the present inventionhas a residual water content of a maximum of 10%, preferably less than5% and more preferably less than 2%, determined according to KarlFischer. Preferably, the pharmaceutical composition of the presentinvention is dried by lyophilization.

The term “drug” as used herein refers to a substance used in thetreatment, cure, prevention, or diagnosis of a disease or used tootherwise enhance physical or mental well-being. If a drug is conjugatedto another moiety, the moiety of the resulting product that originatedfrom the drug is referred to as “biologically active moiety”.

As used herein the term “prodrug” refers to a biologically active moietyreversibly and covalently connected to a specialized protective groupthrough a reversible prodrug linker moiety which is a linker moietycomprising a reversible linkage with the biologically active moiety andwherein the specialized protective group alters or eliminatesundesirable properties in the parent molecule. This also includes theenhancement of desirable properties in the drug and the suppression ofundesirable properties. The specialized non-toxic protective group isreferred to as “carrier”. A prodrug releases the reversibly andcovalently bound biologically active moiety in the form of itscorresponding drug. In other words, a prodrug is a conjugate comprisinga biologically active moiety which is covalently and reversiblyconjugated to a carrier moiety via a reversible prodrug linker moiety,which covalent and reversible conjugation of the carrier to thereversible prodrug linker moiety is either directly or through a spacer.Such conjugate releases the formerly conjugated biologically activemoiety in the form of a free drug.

A “biodegradable linkage” or a “reversible linkage” is a linkage that ishydrolytically degradable, i.e. cleavable, in the absence of enzymesunder physiological conditions (aqueous buffer at pH 7.4, 37° C.) with ahalf-life ranging from one hour to six months, preferably from one hourto four months, even more preferably from one hour to three months, evenmore preferably from one hour to two months, even more preferably fromone hour to one month.

Accordingly, a stable linkage is a linkage having a half-life underphysiological conditions (aqueous buffer at pH 7.4, 37° C.) of more thansix months.

Accordingly, a “reversible prodrug linker moiety” is a moiety which iscovalently conjugated to a biologically active moiety, such as a CNPagonist moiety, through a reversible linkage and is also covalentlyconjugated to a carrier moiety, such as —Z or —Z′, wherein the covalentconjugation to said carrier moiety is either directly or through aspacer moiety, such as -L²-.

Preferably the linkage between —Z or —Z′ and -L²- is a stable linkage.

As used herein, the term “traceless prodrug linker” means a reversibleprodrug linker which upon cleavage releases the drug in its free form.As used herein, the term “free form” of a drug means the drug in itsunmodified, pharmacologically active form.

As used herein, the term “excipient” refers to a diluent, adjuvant, orvehicle with which the therapeutic, such as a drug or prodrug, isadministered. Such pharmaceutical excipient can be sterile liquids, suchas water and oils, including those of petroleum, animal, vegetable orsynthetic origin, including but not limited to peanut oil, soybean oil,mineral oil, sesame oil and the like. Water is a preferred excipientwhen the pharmaceutical composition is administered orally. Saline andaqueous dextrose are preferred excipients when the pharmaceuticalcomposition is administered intravenously. Saline solutions and aqueousdextrose and glycerol solutions are preferably employed as liquidexcipients for injectable solutions. Suitable pharmaceutical excipientsinclude starch, glucose, lactose, sucrose, mannitol, trehalose, gelatin,malt, rice, flour, chalk, silica gel, sodium stearate, glycerolmonostearate, talc, sodium chloride, dried skim milk, glycerol,propylene, glycol, water, ethanol and the like. The pharmaceuticalcomposition, if desired, can also contain minor amounts of wetting oremulsifying agents, pH buffering agents, like, for example, acetate,succinate, tris, carbonate, phosphate, HEPES(4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), MES(2-(N-morpholino)ethanesulfonic acid), or can contain detergents, likeTween, poloxamers, poloxamines, CHAPS, Igepal, or amino acids like, forexample, glycine, lysine, or histidine. These pharmaceuticalcompositions can take the form of solutions, suspensions, emulsions,tablets, pills, capsules, powders, sustained-release formulations andthe like. The pharmaceutical composition can be formulated as asuppository, with traditional binders and excipients such astriglycerides. Oral formulation can include standard excipients such aspharmaceutical grades of mannitol, lactose, starch, magnesium stearate,sodium saccharine, cellulose, magnesium carbonate, etc. Suchcompositions will contain a therapeutically effective amount of the drugor biologically active moiety, together with a suitable amount ofexcipient so as to provide the form for proper administration to thepatient. The formulation should suit the mode of administration.

As used herein, the term “reagent” means a chemical compound whichcomprises at least one functional group for reaction with the functionalgroup of another chemical compound or drug. It is understood that a drugcomprising a functional group (such as a primary or secondary amine orhydroxyl functional group) is also a reagent.

As used herein, the term “moiety” means a part of a molecule, whichlacks one or more atom(s) compared to the corresponding reagent. If, forexample, a reagent of the formula “H—X—H” reacts with another reagentand becomes part of the reaction product, the corresponding moiety ofthe reaction product has the structure “H—X—” or “—X—”, whereas each “—”indicates attachment to another moiety. Accordingly, a biologicallyactive moiety is released from a prodrug as a drug.

It is understood that if the sequence or chemical structure of a groupof atoms is provided which group of atoms is attached to two moieties oris interrupting a moiety, said sequence or chemical structure can beattached to the two moieties in either orientation, unless explicitlystated otherwise. For example, a moiety “—C(O)N(R¹)—” can be attached totwo moieties or interrupting a moiety either as “—C(O)N(R¹)—” or as“—N(R¹)C(O)—”. Similarly, a moiety

can be attached to two moieties or can interrupt a moiety either as

As used herein, the term “functional group” means a group of atoms whichcan react with other groups of atoms. Functional groups include but arenot limited to the following groups: carboxylic acid (—(C═O)OH), primaryor secondary amine (—NH₂, —NH—), maleimide, thiol (—SH), sulfonic acid(—(O═S═O)OH), carbonate, carbamate (—O(C═O)N<), hydroxyl (—OH), aldehyde(—(C═O)H), ketone (—(C═O)—), hydrazine (>N—N<), isocyanate,isothiocyanate, phosphoric acid (—O(P═O)OHOH), phosphonic acid(—O(P═O)OHH), haloacetyl, alkyl halide, acryloyl, aryl fluoride,hydroxylamine, disulfide, sulfonamides, sulfuric acid, vinyl sulfone,vinyl ketone, diazoalkane, oxirane, and aziridine.

In case the controlled-release CNP agonists of the present inventioncomprise one or more acidic or basic groups, the invention alsocomprises their corresponding pharmaceutically or toxicologicallyacceptable salts, in particular their pharmaceutically utilizable salts.Thus, the controlled-release CNP agonists of the present inventioncomprising acidic groups can be used according to the invention, forexample, as alkali metal salts, alkaline earth metal salts or asammonium salts. More precise examples of such salts include sodiumsalts, potassium salts, calcium salts, magnesium salts or salts withammonia or organic amines such as, for example, ethylamine,ethanolamine, triethanolamine or amino acids. Controlled-release CNPagonists of the present invention comprising one or more basic groups,i.e. groups which can be protonated, can be present and can be usedaccording to the invention in the form of their addition salts withinorganic or organic acids. Examples for suitable acids include hydrogenchloride, hydrogen bromide, phosphoric acid, sulfuric acid, nitric acid,methanesulfonic acid, p-toluenesulfonic acid, naphthalenedisulfonicacids, oxalic acid, acetic acid, tartaric acid, lactic acid, salicylicacid, benzoic acid, formic acid, propionic acid, pivalic acid,diethylacetic acid, malonic acid, succinic acid, pimelic acid, fumaricacid, maleic acid, malic acid, sulfaminic acid, phenylpropionic acid,gluconic acid, ascorbic acid, isonicotinic acid, citric acid, adipicacid, and other acids known to the person skilled in the art. For theperson skilled in the art further methods are known for converting thebasic group into a cation like the alkylation of an amine groupresulting in a positively-charge ammonium group and an appropriatecounterion of the salt. If the controlled-release CNP agonists of thepresent invention simultaneously comprise acidic and basic groups, theinvention also includes, in addition to the salt forms mentioned, innersalts or betaines (zwitterions). The respective salts can be obtained bycustomary methods which are known to the person skilled in the art like,for example by contacting these prodrugs with an organic or inorganicacid or base in a solvent or dispersant, or by anion exchange or cationexchange with other salts. The present invention also includes all saltsof the prodrugs of the present invention which, owing to lowphysiological compatibility, are not directly suitable for use inpharmaceuticals but which can be used, for example, as intermediates forchemical reactions or for the preparation of pharmaceutically acceptablesalts.

The term “pharmaceutically acceptable” means a substance that does causeharm when administered to a patient and preferably means approved by aregulatory agency, such as the EMA (Europe) and/or the FDA (US) and/orany other national regulatory agency for use in animals, preferably foruse in humans.

As used herein the term “about” in combination with a numerical value isused to indicate a range ranging from and including the numerical valueplus and minus no more than 10% of said numerical value, more preferablyno more than 8% of said numerical value, even more preferably no morethan 5% of said numerical value and most preferably no more than 2% ofsaid numerical value. For example, the phrase “about 200” is used tomean a range ranging from and including 200+/−10%, i.e. ranging from andincluding 180 to 220; preferably 200+/−8%, i.e. ranging from andincluding 184 to 216; even more preferably ranging from and including200+/−5%, i.e. ranging from and including 190 to 210; and mostpreferably 200+/−2%, i.e. ranging from and including 196 to 204. It isunderstood that a percentage given as “about 20%” does not mean“20%+/−₁₀%”, i.e. ranging from and including 10 to 30%, but “about 20%”means ranging from and including 18 to 22%, i.e. plus and minus 10% ofthe numerical value which is 20.

As used herein, the term “polymer” means a molecule comprising repeatingstructural units, i.e. the monomers, connected by chemical bonds in alinear, circular, branched, crosslinked or dendrimeric way or acombination thereof, which may be of synthetic or biological origin or acombination of both. It is understood that a polymer may also compriseone or more other chemical groups and/or moieties, such as, for example,one or more functional groups. Preferably, a soluble polymer has amolecular weight of at least 0.5 kDa, e.g. a molecular weight of atleast 1 kDa, a molecular weight of at least 2 kDa, a molecular weight ofat least 3 kDa or a molecular weight of at least 5 kDa. If the polymeris soluble, it preferable has a molecular weight of at most 1000 kDa,such as at most 750 kDa, such as at most 500 kDa, such as at most 300kDa, such as at most 200 kDa, such as at most 100 kDa. It is understoodthat for insoluble polymers, such as hydrogels, no meaningful molecularweight ranges can be provided. It is understood that also a protein is apolymer in which the amino acids are the repeating structural units,even though the side chains of each amino acid may be different.

As used herein, the term “polymeric” means a reagent or a moietycomprising one or more polymers or polymer moieties. A polymeric reagentor moiety may optionally also comprise one or more othermoiety/moieties, which are preferably selected from the group consistingof:

-   -   C₁₋₅₀ alkyl, C₂₋₅₀ alkenyl, C₂₋₅₀ alkynyl, C₃₋₁₀ cycloalkyl, 3-        to 10-membered heterocyclyl, 8- to 11-membered heterobicyclyl,        phenyl, naphthyl, indenyl, indanyl, and tetralinyl; and    -   linkages selected from the group comprising

-   -   wherein    -   dashed lines indicate attachment to the remainder of the moiety        or reagent, and    -   —R and —R^(a) are independently of each other selected from the        group consisting of —H, methyl, ethyl, propyl, butyl, pentyl and        hexyl.

The person skilled in the art understands that the polymerizationproducts obtained from a polymerization reaction do not all have thesame molecular weight, but rather exhibit a molecular weightdistribution. Consequently, the molecular weight ranges, molecularweights, ranges of numbers of monomers in a polymer and numbers ofmonomers in a polymer as used herein, refer to the number averagemolecular weight and number average of monomers, i.e. to the arithmeticmean of the molecular weight of the polymer or polymeric moiety and thearithmetic mean of the number of monomers of the polymer or polymericmoiety.

Accordingly, in a polymeric moiety comprising “x” monomer units anyinteger given for “x” therefore corresponds to the arithmetic meannumber of monomers. Any range of integers given for “x” provides therange of integers in which the arithmetic mean numbers of monomers lies.An integer for “x” given as “about x” means that the arithmetic meannumbers of monomers lies in a range of integers of x+/−10%, preferablyx+/−8%, more preferably x+/−5% and most preferably x+/−2%.

As used herein, the term “number average molecular weight” means theordinary arithmetic mean of the molecular weights of the individualpolymers.

As used herein the term “water-soluble” with reference to a carriermeans that when such carrier is part of the controlled-release CNPagonists of the present invention at least 1 g of the controlled-releaseCNP agonists comprising such water-soluble carrier can be dissolved inone liter of water at 20° C. to form a homogeneous solution.Accordingly, the term “water-insoluble” with reference to a carriermeans that when such carrier is part of a controlled-release CNPagonists of the present invention less than 1 g of thecontrolled-release CNP agonists comprising such water-insoluble carriercan be dissolved in one liter of water at 20° C. to form a homogeneoussolution.

As used herein, the term “hydrogel” means a hydrophilic or amphiphilicpolymeric network composed of homopolymers or copolymers, which isinsoluble due to the presence of covalent chemical crosslinks. Thecrosslinks provide the network structure and physical integrity.

As used herein the term “thermogelling” means a compound that is aliquid or a low viscosity solution having a viscosity of less than 500cps at 25° C. at a shear rate of about 0.1/second at a low temperature,which low temperature ranges between about 0° C. to about 10° C., butwhich is a higher viscosity compound of less than 10000 cps at 25° C. ata shear rate of about 0.1/second at a higher temperature, which highertemperature ranges between about 30° C. to about 40° C., such as atabout 37° C.

As used herein, the term “PEG-based” in relation to a moiety or reagentmeans that said moiety or reagent comprises PEG. Preferably, a PEG-basedmoiety or reagent comprises at least 10% (w/w) PEG, such as at least 20%(w/w) PEG, such as at least 30% (w/w) PEG, such as at least 40% (w/w)PEG, such as at least 50% (w/w), such as at least 60 (w/w) PEG, such asat least 70% (w/w) PEG, such as at least 80% (w/w) PEG, such as at least90% (w/w) PEG, such as at least 95% (w/w) PEG. The remaining weightpercentage of the PEG-based moiety or reagent are other moietiespreferably selected from the following moieties and linkages:

-   -   C₁₋₅₀ alkyl, C₂₋₅₀ alkenyl, C₂₋₅₀ alkynyl, C₃₋₁₀ cycloalkyl, 3-        to 10-membered heterocyclyl, 8- to 11-membered heterobicyclyl,        phenyl, naphthyl, indenyl, indanyl, and tetralinyl; and    -   linkages selected from the group comprising

-   -   wherein    -   dashed lines indicate attachment to the remainder of the moiety        or reagent, and    -   —R and —R^(a) are independently of each other selected from the        group consisting of —H, methyl, ethyl, propyl, butyl, pentyl and        hexyl.

As used herein, the term “PEG-based comprising at least X % PEG” inrelation to a moiety or reagent means that said moiety or reagentcomprises at least X % (w/w) ethylene glycol units (—CH₂CH₂O—), whereinthe ethylene glycol units may be arranged blockwise, alternating or maybe randomly distributed within the moiety or reagent and preferably allethylene glycol units of said moiety or reagent are present in oneblock; the remaining weight percentage of the PEG-based moiety orreagent are other moieties preferably selected from the followingmoieties and linkages:

-   -   C₁₋₅₀ alkyl, C₂₋₅₀ alkenyl, C₂₋₅₀ alkynyl, C₃₋₁₀ cycloalkyl, 3-        to 10-membered heterocyclyl, 8- to 11-membered heterobicyclyl,        phenyl, naphthyl, indenyl, indanyl, and tetralinyl; and    -   linkages selected from the group comprising

-   -   wherein    -   dashed lines indicate attachment to the remainder of the moiety        or reagent, and    -   —R and —R^(a) are independently of each other selected from the        group consisting of —H, methyl, ethyl, propyl, butyl, pentyl and        hexyl.

The term “hyaluronic acid-based comprising at least X % hyaluronic acid”is used accordingly.

The term “substituted” as used herein means that one or more —H atom(s)of a molecule or moiety are replaced by a different atom or a group ofatoms, which are referred to as “substituent”.

Preferably, the one or more further optional substituents areindependently of each other selected from the group consisting ofhalogen, —CN, —COOR^(x1), —OR^(x1), —C(O)R^(x1), —C(O)N(R^(x1)R^(x1a)),—S(O)₂N(R_(x1)R^(x1a)), —S(O)N(R^(x1)R_(x1a)), —S(O)₂R^(x1),—S(O)R^(x1), —N(R^(x1))S(O)₂N(R^(x1a)R^(x1b)), —SR^(x1),—N(R^(x1)R^(x1a)), —NO₂, —OC(O)R^(x1), —N(R^(x1))C(O)R^(x1a),—N(R^(x1))S(O)₂R^(x1a), —N(R^(x1))S(O)R^(x1a), —N(R^(x1))C(O)OR^(x1a),—N(R^(x1))C(O)N(R^(x1a)R^(x1b)), —OC(O)N(R^(x1)R^(x1a)), -T⁰, C₁₋₅₀alkyl, C₂₋₅₀ alkenyl, and C₂₋₅₀ alkynyl; wherein -T⁰, C₁₋₅₀ alkyl, C₂₋₅₀alkenyl, and C₂₋₅₀ alkynyl are optionally substituted with one or more—R^(x2), which are the same or different and wherein C₁₋₅₀ alkyl, C₂₋₅₀alkenyl, and C₂₋₅₀ alkynyl are optionally interrupted by one or moregroups selected from the group consisting of -T⁰-, —C(O)O—, —O—, —C(O)—,—C(O)N(R^(x3))—, —S(O)₂N(R^(x3))—, —S(O)N(R^(x3))—, —S(O)₂—, —S(O)—,—N(R^(x3))S(O)₂N(R^(x3a))—S—, —N(R^(x3))—, —OC(OR^(x3))(R^(x3a))—,—N(R^(x3))C(O)N(R^(x3a))—, and —OC(O)N(R^(x3))—;

—R^(x1), —R^(x1a), —R^(x1b) are independently of each other selectedfrom the group consisting of —H, -T⁰, C₁₋₅₀ alkyl, C₂₋₅₀ alkenyl, andC₂₋₅₀ alkynyl; wherein -T⁰, C₁₋₅₀ alkyl, C₂₋₅₀ alkenyl, and C₂₋₅₀alkynyl are optionally substituted with one or more —R_(x2), which arethe same or different and wherein C₁₋₅₀ alkyl, C₂₋₅₀ alkenyl, and C₂₋₅₀alkynyl are optionally interrupted by one or more groups selected fromthe group consisting of -T⁰-, —C(O)O—, —O—, —C(O)—, —C(O)N(R^(x3))—,—S(O)₂N(R^(x3))—, —S(O)N(R^(x3))—; —S(O)₂—, —S(O)—,—N(R^(x3))S(O)₂N(R^(x3a))—, —S—, —N(R^(x3))—, —OC(OR^(x3))(R^(x3a))—,—N(R^(x3))C(O)N(R^(x3a))—, and —OC(O)N(R^(x3))—;

each T⁰ is independently selected from the group consisting of phenyl,naphthyl, indenyl, indanyl, tetralinyl, C₃₋₁₀ cycloalkyl, 3- to10-membered heterocyclyl, and 8- to 11-membered heterobicyclyl; whereineach T₀ is independently optionally substituted with one or more —R^(x2)which are the same or different;

each —R^(x2) is independently selected from the group consisting ofhalogen, —CN, oxo (═O), —COOR^(x4), —OR^(x4), —C(O)R^(x4),—C(O)N(R^(x4)R^(x4a)), —S(O)₂N(R^(x4)R^(x4a)), —S(O)N(R^(x4)R^(x4a)),—S(O)₂R^(x4), —S(O)R^(x4), —N(R^(x4))S(O)₂N(R^(x4a)R^(x4b)), —SR^(x4),—N(R^(x4)R^(x4a)), —NO₂, —OC(O)R^(x4), —N(R^(x4))C(O)R^(x4a),—N(R^(x4))S(O)₂R^(x4a), —N(R^(x4))S(O)R^(x4a), —N(R^(x4))C(O)OR^(x4a),—N(R^(x4))C(O)N(R^(x4a)R^(x4b)), —OC(O)N(R^(x4)R^(x4a)), and C₁₋₆ alkyl;wherein C₁₋₆ alkyl is optionally substituted with one or more halogen,which are the same or different;

each —R^(x3), —R^(x3a), —R^(x4), —R^(x4a), —R^(x4b) is independentlyselected from the group consisting of —H and C₁₋₆ alkyl; wherein C₁₋₆alkyl is optionally substituted with one or more halogen, which are thesame or different.

More preferably, the one or more further optional substituents areindependently of each other selected from the group consisting ofhalogen, —CN, —COOR^(x1), —OR^(x1), —C(O)R^(x1), —C(O)N(R^(x1)R^(x1a)),—S(O)₂N(R^(x1)R^(x1a)), —S(O)N(R^(x1)R^(x1a)), —S(O)₂R^(x1),—S(O)R^(x1), —N(R^(x1))S(O)₂N(R^(x1a)R^(x1b)), —SR^(x1),—N(R^(x1)R^(x1a)), —NO₂, —OC(O)R^(x1), —N(R^(x1))C(O)R^(x1a),—N(R^(x1))S(O)₂R^(x1a), —N(R^(x1))S(O)R^(x1a), —N(R^(x1))C(O)OR^(x1a),—N(R^(x1))C(O)N(R^(x1a)R^(x1b)), —OC(O)N(R^(x1)R^(x1a)), -T⁰, C₁₋₁₀alkyl, C₂₋₁₀ alkenyl, and C₂₋₁₀ alkynyl; wherein -T⁰-, C₁₋₁₀ alkyl,C₂₋₁₀ alkenyl, and C₂₋₁₀ alkynyl are optionally substituted with one ormore —R^(x2), which are the same or different and wherein C₁₋₁₀ alkyl,C₂₋₁₀ alkenyl, and C₂₋₁₀ alkynyl are optionally interrupted by one ormore groups selected from the group consisting of -T⁰-, —C(O)O—, —O—,—C(O)—, —C(O)N(R^(x3))—, —S(O)₂N(R^(x3))—, —S(O)N(R^(x3))—, —S(O)₂—,—S(O)—, —N(R^(x3))S(O)₂N(R^(x3a))—, —S—, —N(R^(x3))—,—OC(OR^(x3))(R^(x3a))—, —N(R^(x3))C(O)N(R^(x3a))—, and —OC(O)N(R^(x3))—;

each —R^(x1), —R^(x1a), —R^(x1b), —R^(x3), —R^(x3a) is independentlyselected from the group consisting of —H, halogen, C₁₋₆ alkyl, C₂₋₆alkenyl, and C₂₋₆ alkynyl;

each T⁰ is independently selected from the group consisting of phenyl,naphthyl, indenyl, indanyl, tetralinyl, C₃₋₁₀ cycloalkyl, 3- to10-membered heterocyclyl, and 8- to 11-membered heterobicyclyl; whereineach T⁰ is independently optionally substituted with one or more—R^(x2), which are the same or different;

each —R^(x2) is independently selected from the group consisting ofhalogen, —CN, oxo (═O), —COOR^(x4), —OR^(x4), —C(O)R^(x4),—C(O)N(R^(x4)R^(x4a)), —S(O)₂N(R^(x4)R^(x4a)), —S(O)N(R^(x4)R^(x4a)),—S(O)₂R^(x4), —S(O)R^(x4), —N(R^(x4))S(O)₂N(R^(x4a)R^(x4b)), —SR^(x4),—N(R^(x4)R^(x4a)), —NO₂, —OC(O)R^(x4), —N(R^(x4))C(O)R^(x4)a,—N(R^(x4))S(O)₂R^(x4a), —N(R^(x4))S(O)R^(x4)a, —N(R^(x4))C(O)OR^(x4a),—N(R^(x4))C(O)N(R^(x4a)R^(x4b)), —OC(O)N(R^(x4)R^(x4a)), and C₁₋₆ alkyl;wherein C₁₋₆ alkyl is optionally substituted with one or more halogen,which are the same or different;

each —R^(x4), —R^(x4a), —R^(x4b) is independently selected from thegroup consisting of —H, halogen, C₁₋₆ alkyl, C₂₋₆ alkenyl, and C₂₋₆alkynyl;

Even more preferably, the one or more further optional substituents areindependently of each other selected from the group consisting ofhalogen, —CN, —COOR^(x1), —OR^(x1), —C(O)R^(x1), —C(O)N(R^(x1)R^(x1a)),—S(O)₂N(R^(x1)R^(x1a)), —S(O)N(R^(x1)R^(x1a)), —S(O)₂R^(x1),—S(O)R^(x1), —N(R^(x1))S(O)₂N(R^(x1a)R^(x1b)), —SR^(x1),—N(R^(x1)R^(x1a)), —NO₂, —OC(O)R^(x1), —N(R^(x1))C(O)R^(x1a),—N(R^(x1))S(O)₂R^(x1a), —N(R^(x1))S(O)R^(x1a), —N(R^(x1))C(O)OR^(x1a),—N(R^(x1))C(O)N(R^(x1a)R^(x1b)), —OC(O)N(R^(x1)R^(x1a)), -T⁰, C₁₋₆alkyl, C₂₋₆ alkenyl, and C₂₋₆ alkynyl; wherein -T⁰, C₁₋₆ alkyl, C₂₋₆alkenyl, and C₂₋₆ alkynyl are optionally substituted with one or more—R^(x2), which are the same or different and wherein C₁₋₆ alkyl, C₂₋₆alkenyl, and C₂₋₆ alkynyl are optionally interrupted by one or moregroups selected from the group consisting of -T⁰, —C(O)O—, —O—, —C(O)—,—C(O)N(R^(x3))—, —S(O)₂N(R^(x3))—, —S(O)N(R^(x3))—, —S(O)₂—, —S(O)—,—N(R^(x3))S(O)₂N(R^(x3a))—, —S—, —N(R^(x3))—, —OC(OR^(x3))(R^(x3a))—,—N(R^(x3))C(O)N(R^(x3a))—, and —OC(O)N(R^(x3))—;

each —R^(x1), —R^(x1a), —R^(x1b), —R^(x2), —R^(x3), —R^(x3a) isindependently selected from the group consisting of —H, halogen, C₁₋₆alkyl, C₂₋₆ alkenyl, and C₂₋₆ alkynyl;

each T⁰ is independently selected from the group consisting of phenyl,naphthyl, indenyl, indanyl, tetralinyl, C₃₋₁₀ cycloalkyl, 3- to10-membered heterocyclyl, and 8- to 11-membered heterobicyclyl; whereineach T⁰ is independently optionally substituted with one or more—R^(x2), which are the same or different.

Preferably, a maximum of 6-H atoms of an optionally substituted moleculeare independently replaced by a substituent, e.g. 5-H atoms areindependently replaced by a substituent, 4-H atoms are independentlyreplaced by a substituent, 3-H atoms are independently replaced by asubstituent, 2-H atoms are independently replaced by a substituent, or1-H atom is replaced by a substituent.

The term “interrupted” means that a moiety is inserted between twocarbon atoms or—if the insertion is at one of the moiety's ends—betweena carbon or heteroatom and a hydrogen atom, preferably between a carbonand a hydrogen atom.

As used herein, the term “C₁₋₄ alkyl” alone or in combination means astraight-chain or branched alkyl moiety having 1 to 4 carbon atoms. Ifpresent at the end of a molecule, examples of straight-chain or branchedC₁₋₄ alkyl are methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl,sec-butyl and tert-butyl. When two moieties of a molecule are linked bythe C₁₋₄ alkyl, then examples for such C₁₋₄ alkyl groups are —CH₂—,—CH₂—CH₂—, —CH(CH₃)—, —CH₂—CH₂—CH₂—, —CH(C₂H₅)—, —C(CH₃)₂—. Eachhydrogen of a C₁₋₄ alkyl carbon may optionally be replaced by asubstituent as defined above. Optionally, a C₁₋₄ alkyl may beinterrupted by one or more moieties as defined below.

As used herein, the term “C₁₋₆ alkyl” alone or in combination means astraight-chain or branched alkyl moiety having 1 to 6 carbon atoms. Ifpresent at the end of a molecule, examples of straight-chain andbranched C₁₋₆ alkyl groups are methyl, ethyl, n-propyl, isopropyl,n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, 2-methylbutyl,2,2-dimethylpropyl, n-hexyl, 2-methylpentyl, 3-methylpentyl,2,2-dimethylbutyl, 2,3-dimethylbutyl and 3,3-dimethylpropyl. When twomoieties of a molecule are linked by the C₁₋₆ alkyl group, then examplesfor such C₁₋₆ alkyl groups are —CH₂—, —CH₂—CH₂—, —CH(CH₃)—,—CH₂—CH₂—CH₂—, —CH(C₂H₅)— and —C(CH₃)₂—. Each hydrogen atom of a C₁₋₆carbon may optionally be replaced by a substituent as defined above.Optionally, a C₁₋₆ alkyl may be interrupted by one or more moieties asdefined below.

Accordingly, “C₁₋₁₀ alkyl”, “C₁₋₂₀ alkyl” or “C₁₋₅₀ alkyl” means analkyl chain having 1 to 10, 1 to 20 or 1 to 50 carbon atoms,respectively, wherein each hydrogen atom of the C₁₋₁₀, C₁₋₂₀ or C₁₋₅₀carbon may optionally be replaced by a substituent as defined above.Optionally, a C₁₋₁₀ or C₁₋₅₀ alkyl may be interrupted by one or moremoieties as defined below.

As used herein, the term “C₂₋₆ alkenyl” alone or in combination means astraight-chain or branched hydrocarbon moiety comprising at least onecarbon-carbon double bond having 2 to 6 carbon atoms. If present at theend of a molecule, examples are —CH═CH₂, —CH═CH—CH₃, —CH₂—CH═CH₂,—CH═CHCH₂—CH₃ and —CH═CH—CH═CH₂. When two moieties of a molecule arelinked by the C₂₋₆ alkenyl group, then an example for such C₂₋₆ alkenylis —CH═CH—. Each hydrogen atom of a C₂₋₆ alkenyl moiety may optionallybe replaced by a substituent as defined above. Optionally, a C₂₋₆alkenyl may be interrupted by one or more moieties as defined below.

Accordingly, the term “C₂₋₁₀ alkenyl”, “C₂₋₂₀ alkenyl” or “C₂₋₅₀alkenyl” alone or in combination means a straight-chain or branchedhydrocarbon moiety comprising at least one carbon-carbon double bondhaving 2 to 10, 2 to 20 or 2 to 50 carbon atoms. Each hydrogen atom of aC₂₋₁₀ alkenyl, C₂₋₂₀ alkenyl or C₂₋₅₀ alkenyl group may optionally bereplaced by a substituent as defined above. Optionally, a C₂₋₁₀ alkenyl,C₂₋₂₀ alkenyl or C₂₋₅₀ alkenyl may be interrupted by one or moremoieties as defined below.

As used herein, the term “C₂₋₆ alkynyl” alone or in combination means astraight-chain or branched hydrocarbon moiety comprising at least onecarbon-carbon triple bond having 2 to 6 carbon atoms. If present at theend of a molecule, examples are —C≡CH, —CH₂—C≡CH, CH₂—CH₂—C≡CH andCH₂—C≡C—CH₃. When two moieties of a molecule are linked by the alkynylgroup, then an example is —C≡C—. Each hydrogen atom of a C₂₋₆ alkynylgroup may optionally be replaced by a substituent as defined above.Optionally, one or more double bond(s) may occur. Optionally, a C₂₋₆alkynyl may be interrupted by one or more moieties as defined below.

Accordingly, as used herein, the term “C₂₋₁₀ alkynyl”, “C₂₋₂₀ alkynyl”and “C₂₋₅₀ alkynyl” alone or in combination means a straight-chain orbranched hydrocarbon moiety comprising at least one carbon-carbon triplebond having 2 to 10, 2 to 20 or 2 to 50 carbon atoms, respectively. Eachhydrogen atom of a C₂₋₁₀ alkynyl, C₂₋₂₀ alkynyl or C₂₋₅₀ alkynyl groupmay optionally be replaced by a substituent as defined above.Optionally, one or more double bond(s) may occur. Optionally, a C₂₋₁₀alkynyl, C₂₋₂₀ alkynyl or C₂₋₅₀ alkynyl may be interrupted by one ormore moieties as defined below.

As mentioned above, a C₁₋₄ alkyl, C₁₋₆ alkyl, C₁₋₁₀ alkyl, C₁₋₂₀ alkyl,C₁₋₅₀ alkyl, C₂₋₆ alkenyl, C₂₋₁₀ alkenyl, C₂₋₂₀ alkenyl, C₂₋₅₀ alkenyl,C₂₋₆ alkynyl, C₂₋₁₀ alkynyl, C₂₋₂₀ alkenyl or C₂₋₅ alkynyl mayoptionally be interrupted by one or more moieties which are preferablyselected from the group consisting of

-   -   wherein    -   dashed lines indicate attachment to the remainder of the moiety        or reagent; and    -   —R and —R^(a) are independently of each other selected from the        group consisting of —H, methyl, ethyl, propyl, butyl, pentyl and        hexyl.

As used herein, the term “C₃₋₁₀ cycloalkyl” means a cyclic alkyl chainhaving 3 to 10 carbon atoms, which may be saturated or unsaturated, e.g.cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cyclohexenyl,cycloheptyl, cyclooctyl, cyclononyl or cyclodecyl. Each hydrogen atom ofa C₃₋₁₀ cycloalkyl carbon may be replaced by a substituent as definedabove. The term “C₃₋₁₀ cycloalkyl” also includes bridged bicycles likenorbornane or norbornene.

The term “8- to 30-membered carbopolycyclyl” or “8- to 30-memberedcarbopolycycle” means a cyclic moiety of two or more rings with 8 to 30ring atoms, where two neighboring rings share at least one ring atom andthat may contain up to the maximum number of double bonds (aromatic ornon-aromatic ring which is fully, partially or un-saturated). Preferablya 8- to 30-membered carbopolycyclyl means a cyclic moiety of two, three,four or five rings, more preferably of two, three or four rings.

As used herein, the term “3- to 10-membered heterocyclyl” or “3- to10-membered heterocycle” means a ring with 3, 4, 5, 6, 7, 8, 9 or 10ring atoms that may contain up to the maximum number of double bonds(aromatic or non-aromatic ring which is fully, partially orun-saturated) wherein at least one ring atom up to 4 ring atoms arereplaced by a heteroatom selected from the group consisting of sulfur(including —S(O)—, —S(O)₂—), oxygen and nitrogen (including ═N(O)—) andwherein the ring is linked to the rest of the molecule via a carbon ornitrogen atom. Examples for 3- to 10-membered heterocycles include butare not limited to aziridine, oxirane, thiirane, azirine, oxirene,thiirene, azetidine, oxetane, thietane, furan, thiophene, pyrrole,pyrroline, imidazole, imidazoline, pyrazole, pyrazoline, oxazole,oxazoline, isoxazole, isoxazoline, thiazole, thiazoline, isothiazole,isothiazoline, thiadiazole, thiadiazoline, tetrahydrofuran,tetrahydrothiophene, pyrrolidine, imidazolidine, pyrazolidine,oxazolidine, isoxazolidine, thiazolidine, isothiazolidine,thiadiazolidine, sulfolane, pyran, dihydropyran, tetrahydropyran,imidazolidine, pyridine, pyridazine, pyrazine, pyrimidine, piperazine,piperidine, morpholine, tetrazole, triazole, triazolidine,tetrazolidine, diazepane, azepine and homopiperazine. Each hydrogen atomof a 3- to 10-membered heterocyclyl or 3- to 10-membered heterocyclicgroup may be replaced by a substituent as defined below.

As used herein, the term “8- to 11-membered heterobicyclyl” or “8- to11-membered heterobicycle” means a heterocyclic moiety of two rings with8 to 11 ring atoms, where at least one ring atom is shared by both ringsand that may contain up to the maximum number of double bonds (aromaticor non-aromatic ring which is fully, partially or un-saturated) whereinat least one ring atom up to 6 ring atoms are replaced by a heteroatomselected from the group consisting of sulfur (including —S(O)—,—S(O)₂—), oxygen and nitrogen (including ═N(O)—) and wherein the ring islinked to the rest of the molecule via a carbon or nitrogen atom.Examples for an 8- to 11-membered heterobicycle are indole, indoline,benzofuran, benzothiophene, benzoxazole, benzisoxazole, benzothiazole,benzisothiazole, benzimidazole, benzimidazoline, quinoline, quinazoline,dihydroquinazoline, quinoline, dihydroquinoline, tetrahydroquino line,decahydroquino line, isoquinoline, decahydroisoquino line,tetrahydroisoquinoline, dihydroisoquinoline, benzazepine, purine andpteridine. The term 8- to 11-membered heterobicycle also includes spirostructures of two rings like 1,4-dioxa-8-azaspiro[4.5]decane or bridgedheterocycles like 8-aza-bicyclo[3.2.1]octane. Each hydrogen atom of an8- to 11-membered heterobicyclyl or 8- to 11-membered heterobicyclecarbon may be replaced by a substituent as defined below.

Similarly, the term “8- to 30-membered heteropolycyclyl” or “8- to30-membered heteropolycycle” means a heterocyclic moiety of more thantwo rings with 8 to 30 ring atoms, preferably of three, four or fiverings, where two neighboring rings share at least one ring atom and thatmay contain up to the maximum number of double bonds (aromatic ornon-aromatic ring which is fully, partially or unsaturated), wherein atleast one ring atom up to 10 ring atoms are replaced by a heteroatomselected from the group of sulfur (including —S(O)—, —S(O)₂—), oxygenand nitrogen (including ═N(O)—) and wherein the ring is linked to therest of a molecule via a carbon or nitrogen atom.

It is understood that the phrase “the pair R^(X)/R^(y) is joinedtogether with the atom to which they are attached to form a C₃₋₁₀cycloalkyl or a 3- to 10-membered heterocyclyl” in relation with amoiety of the structure

means that R^(x) and R^(y) form the following structure:

wherein R is C₃₋₁₀ cycloalkyl or 3- to 10-membered heterocyclyl.

It is also understood that the phrase “the pair R^(x)/R^(y) is jointtogether with the atoms to which they are attached to form a ring A” inrelation with a moiety of the structure

means that R^(x) and R^(y) form the following structure:

As used herein, “halogen” means fluoro, chloro, bromo or iodo. It isgenerally preferred that halogen is fluoro or chloro.

In general, the term “comprise” or “comprising” also encompasses“consist of” or “consisting of”.

Preferably the EC₅₀ of the released CNP agonist is measured after therelease of 50% of the CNP agonist comprised in the controlled-releaseCNP agonist.

The controlled-release CNP agonist from which CNP agonist is releasedwith a release half-life of at least 6 hours under physiologicalconditions and which controlled-release CNP agonist has an EC₅₀ that isat least 20-fold higher than the EC₅₀ of the corresponding free CNPagonist and which released CNP agonist has an EC₅₀ that is at most3-fold higher than the EC₅₀ of the corresponding free CNP agonist.

The controlled-release CNP agonist of the present invention releases atleast one CNP agonist under physiological conditions with a releasehalf-life of at least 6 hours. Preferably the controlled-release CNPagonist of the present invention releases at least one CNP agonist underphysiological conditions with a release half-life of at least 12 hours.Even more preferably the controlled-release CNP agonist of the presentinvention releases at least one CNP agonist under physiologicalconditions with a release half-life of at least 24 hours. Even morepreferably the controlled-release CNP agonist of the present inventionreleases at least one CNP agonist under physiological conditions with arelease half-life of at least 48 hours. Even more preferably thecontrolled-release CNP agonist of the present invention releases atleast one CNP agonist under physiological conditions with a releasehalf-life of at least 72 hours. Even more preferably thecontrolled-release CNP agonist of the present invention releases atleast one CNP agonist under physiological conditions with a releasehalf-life of at least 96 hours. Even more preferably thecontrolled-release CNP agonist of the present invention releases atleast one CNP agonist under physiological conditions with a releasehalf-life of at least 120 hours. Even more preferably thecontrolled-release CNP agonist of the present invention releases atleast one CNP agonist under physiological conditions with a releasehalf-life of at least 144 hours. Even more preferably thecontrolled-release CNP agonist of the present invention releases atleast one CNP agonist under physiological conditions with a releasehalf-life of at least 168 hours. Even more preferably thecontrolled-release CNP agonist of the present invention releases atleast one CNP agonist under physiological conditions with a releasehalf-life of at least 192 hours.

The controlled-release CNP agonist has an EC₅₀ that is at least 20-foldhigher than the EC₅₀ of the corresponding free CNP agonist. Preferablythe controlled-release CNP agonist has an EC₅₀ that is at least 30-foldhigher than the EC₅₀ of the corresponding free CNP agonist. Even morepreferably the controlled-release CNP agonist has an EC₅₀ that is atleast 40-fold higher than the EC₅₀ of the corresponding free CNPagonist. Even more preferably the controlled-release CNP agonist has anEC₅₀ that is at least 50-fold higher than the EC₅₀ of the correspondingfree CNP agonist. Even more preferably the controlled-release CNPagonist has an EC₅₀ that is at least 60-fold higher than the EC₅₀ of thecorresponding free CNP agonist. Even more preferably thecontrolled-release CNP agonist has an EC₅₀ that is at least 70-foldhigher than the EC₅₀ of the corresponding free CNP agonist. Even morepreferably the controlled-release CNP agonist has an EC₅₀ that is atleast 80-fold higher than the EC₅₀ of the corresponding free CNPagonist. Even more preferably the controlled-release CNP agonist has anEC₅₀ that is at least 90-fold higher than the EC₅₀ of the correspondingfree CNP agonist. Even more preferably the controlled-release CNPagonist has an EC₅₀ that is at least 100-fold higher than the EC₅₀ ofthe corresponding free CNP agonist. Even more preferably thecontrolled-release CNP agonist has an EC₅₀ that is at least 110-foldhigher than the EC₅₀ of the corresponding free CNP agonist. Even morepreferably the controlled-release CNP agonist has an EC₅₀ that is atleast 120-fold higher than the EC₅₀ of the corresponding free CNPagonist. Even more preferably the controlled-release CNP agonist has anEC₅₀ that is at least 130-fold higher than the EC₅₀ of the correspondingfree CNP agonist. Even more preferably the controlled-release CNPagonist has an EC₅₀ that is at least 140-fold higher than the EC₅₀ ofthe corresponding free CNP agonist. Even more preferably thecontrolled-release CNP agonist has an EC₅₀ that is at least 150-foldhigher than the EC₅₀ of the corresponding free CNP agonist. Even morepreferably the controlled-release CNP agonist has an EC₅₀ that is atleast 160-fold higher than the EC₅₀ of the corresponding free CNPagonist. Even more preferably the controlled-release CNP agonist has anEC₅₀ that is at least 170-fold higher than the EC₅₀ of the correspondingfree CNP agonist. Even more preferably the controlled-release CNPagonist has an EC₅₀ that is at least 180-fold higher than the EC₅₀ ofthe corresponding free CNP agonist. Even more preferably thecontrolled-release CNP agonist has an EC₅₀ that is at least 190-foldhigher than the EC₅₀ of the corresponding free CNP agonist. Even morepreferably the controlled-release CNP agonist has an EC₅₀ that is atleast 200-fold higher than the EC₅₀ of the corresponding free CNPagonist.

The released CNP agonist has an EC₅₀ that is at most 3-fold higher thanthe EC₅₀ of the corresponding free CNP agonist. Preferably the releasedCNP agonist has an EC₅₀ that is at most 2.5-fold higher than the EC₅₀ ofthe corresponding free CNP agonist. Even more preferably the releasedCNP agonist has an EC₅₀ that is at most 2-fold higher than the EC₅₀ ofthe corresponding free CNP agonist. Even more preferably the releasedCNP agonist has an EC₅₀ that is at most 1.5-fold higher than the EC₅₀ ofthe corresponding free CNP agonist.

Even more preferably the released CNP agonist has an EC₅₀ that is atmost 1-fold higher than the EC₅₀ of the corresponding free CNP agonist,i.e. the EC₅₀ is the same for the released CNP agonist and thecorresponding free CNP agonist.

The controlled-release CNP agonist preferably comprises a CNP agonistselected from the group consisting of small molecules, natural products,oligonucleotides, polypeptides and proteins.

In one embodiment the CNP agonist comprises a small molecule.Preferably, the CNP agonist is a small molecule.

In another embodiment the CNP agonist comprises a natural product.Preferably, the CNP agonist is a natural product.

In another embodiment the CNP agonist comprises an oligonucleotide.Preferably, such oligonucleotide is selected from the group consistingof antisense oligonucleotides, aptamers, RNAi and siRNA. Preferably, theCNP agonist is an oligonucleotide, more preferably selected from thegroup consisting of antisense oligonucleotides, aptamers, RNAi andsiRNA.

In another embodiment the CNP agonist comprises a protein. Preferably,the CNP agonist is a protein.

In a preferred embodiment the CNP agonist comprises a polypeptide. Morepreferably the CNP agonist is a polypeptide. Preferably the CNP agonistcomprises a CNP molecule or moiety. More preferably the CNP agonist isCNP. Even more preferably the CNP agonist comprises a CNP molecule ormoiety having the sequence of SEQ ID NO:24, SEQ ID NO:25 or SEQ IDNO:30. Even more preferably the CNP agonist is CNP having the sequenceof SEQ ID NO:24, SEQ ID NO:25 or SEQ ID NO:30. Even more preferably theCNP agonist comprises a CNP molecule or moiety CNP having the sequenceof SEQ ID NO:24. Most preferably the CNP agonist is a CNP having thesequence of SEQ ID NO:24. It is also preferred that the CNP agonist is aCNP having the sequence of SEQ ID NO:20. It is also preferred that theCNP agonist is a CNP having the sequence of SEQ ID NO:21. It is alsopreferred that the CNP agonist is a CNP having the sequence of SEQ IDNO:22. It is also preferred that the CNP agonist is a CNP having thesequence of SEQ ID NO:22. It is also preferred that the CNP agonist is aCNP having the sequence of SEQ ID NO:30.

In one embodiment the controlled-release CNP agonist is water-insoluble.

Preferably, the controlled-release CNP agonist is selected from thegroup consisting of crystals, nanoparticles, microparticles, nanospheresand microspheres.

In one embodiment the controlled-release CNP agonist is a crystalcomprising at least one CNP agonist.

In another embodiment the controlled-release CNP agonist is ananoparticle comprising at least one CNP agonist.

In another embodiment the controlled-release CNP agonist is amicroparticle comprising at least one CNP agonist.

In another embodiment the controlled-release CNP agonist is a nanospherecomprising at least one CNP agonist.

In another embodiment the controlled-release CNP agonist is amicrosphere comprising at least one CNP agonist.

In one embodiment the controlled-release CNP agonist is a vesiclecomprising at least one CNP agonist. Preferably, such vesicle comprisingat least one CNP agonist is a micelle, liposome or polymersome.

In one embodiment the controlled-release CNP agonist is a micellecomprising at least one CNP agonist.

In another embodiment the controlled-release CNP agonist is a liposomecomprising at least one CNP agonist. Preferably, such liposome isselected from the group consisting of aquasomes; non-ionic surfactantvesicles, such as niosomes and proniosomes; cationic liposomes, such asLeciPlex; transfersomes; ethosomes; ufasomes; sphingosomes; andpharmacosomes.

In another embodiment the controlled-release CNP agonist is apolymersome comprising at least one CNP agonist.

In another embodiment the controlled-release CNP agonist comprises atleast one CNP agonist non-covalently embedded in a water-insolublepolymer. Preferably, such water-insoluble polymer comprises a polymerselected from the group consisting of 2-methacryloyl-oxyethyl phosphoylcholins, poly(acrylic acids), poly(acrylates), poly(acrylamides),poly(alkyloxy) polymers, poly(amides), poly(amidoamines), poly(aminoacids), poly(anhydrides), poly(aspartamides), poly(butyric acids),poly(glycolic acids), polybutylene terephthalates, poly(caprolactones),poly(carbonates), poly(cyanoacrylates), poly(dimethylacrylamides),poly(esters), poly(ethylenes), poly(ethyleneglycols), poly(ethyleneoxides), poly(ethyl phosphates), poly(ethyloxazolines), poly(glycolicacids), poly(hydroxyethyl acrylates), poly(hydroxyethyl-oxazolines),poly(hydroxymethacrylates), poly(hydroxypropylmethacrylamides),poly(hydroxypropyl methacrylates), poly(hydroxypropyloxazolines),poly(iminocarbonates), poly(lactic acids), poly(lactic-co-glycolicacids), poly(methacrylamides), poly(methacrylates),poly(methyloxazolines), poly(organophosphazenes), poly(ortho esters),poly(oxazolines), poly(propylene glycols), poly(siloxanes),poly(urethanes), poly(vinyl alcohols), poly(vinyl amines),poly(vinylmethylethers), poly(vinylpyrrolidones), silicones, celluloses,carbomethyl celluloses, hydroxypropyl methylcelluloses, chitins,chitosans, dextrans, dextrins, gelatins, hyaluronic acids andderivatives, functionalized hyaluronic acids, mannans, pectins,rhamnogalacturonans, starches, hydroxyalkyl starches, hydroxyethylstarches and other carbohydrate-based polymers, xylans, and copolymersthereof.

In a preferred embodiment the controlled-release CNP comprises at leastone CNP agonist non-covalently embedded in poly(lactic-co-glycolic acid)(PLGA).

In another embodiment the controlled-release CNP agonist comprises atleast one CNP agonist covalently and reversibly conjugated to awater-insoluble polymer. Preferably such water-insoluble polymercomprises a polymer selected from the group consisting of2-methacryloyl-oxyethyl phosphoyl cholins, poly(acrylic acids),poly(acrylates), poly(acrylamides), poly(alkyloxy) polymers,poly(amides), poly(amidoamines), poly(amino acids), poly(anhydrides),poly(aspartamides), poly(butyric acids), poly(glycolic acids),polybutylene terephthalates, poly(caprolactones), poly(carbonates),poly(cyanoacrylates), poly(dimethylacrylamides), poly(esters),poly(ethylenes), poly(ethyleneglycols), poly(ethylene oxides),poly(ethyl phosphates), poly(ethyloxazolines), poly(glycolic acids),poly(hydroxyethyl acrylates), poly(hydroxyethyl-oxazolines),poly(hydroxymethacrylates), poly(hydroxypropylmethacrylamides),poly(hydroxypropyl methacrylates), poly(hydroxypropyloxazolines),poly(iminocarbonates), poly(lactic acids), poly(lactic-co-glycolicacids), poly(methacrylamides), poly(methacrylates),poly(methyloxazolines), poly(organophosphazenes), poly(ortho esters),poly(oxazolines), poly(propylene glycols), poly(siloxanes),poly(urethanes), poly(vinyl alcohols), poly(vinyl amines),poly(vinylmethylethers), poly(vinylpyrrolidones), silicones, celluloses,carbomethyl celluloses, hydroxypropyl methylcelluloses, chitins,chitosans, dextrans, dextrins, gelatins, hyaluronic acids andderivatives, functionalized hyaluronic acids, mannans, pectins,rhamnogalacturonans, starches, hydroxyalkyl starches, hydroxyethylstarches and other carbohydrate-based polymers, xylans, and copolymersthereof.

Preferably such controlled-release CNP agonist comprising at least oneCNP agonist covalently and reversibly conjugated to a water-insolublepolymer is a CNP agonist prodrug comprising a conjugate D-L, wherein

-   -   -D is a CNP agonist moiety; and    -   -L comprises a reversible prodrug linker moiety -L¹-;    -   wherein -L¹- is substituted with -L²-Z′ and is optionally        further substituted; wherein    -   -L²- is a single chemical bond or a spacer moiety; and    -   —Z′ is a water-insoluble carrier moiety.

It is understood that a multitude of moieties -L²-L¹-D is connected to awater-insoluble carrier —Z′.

The water-insoluble carrier —Z′ is preferably a hydrogel. Preferably,such hydrogel comprises a polymer selected from the group consisting of2-methacryloyl-oxyethyl phosphoyl cholins, poly(acrylic acids),poly(acrylates), poly(acrylamides), poly(alkyloxy) polymers,poly(amides), poly(amidoamines), poly(amino acids), poly(anhydrides),poly(aspartamides), poly(butyric acids), poly(glycolic acids),polybutylene terephthalates, poly(caprolactones), poly(carbonates),poly(cyanoacrylates), poly(dimethylacrylamides), poly(esters),poly(ethylenes), poly(ethyleneglycols), poly(ethylene oxides),poly(ethyl phosphates), poly(ethyloxazolines), poly(glycolic acids),poly(hydroxyethyl acrylates), poly(hydroxyethyl-oxazolines),poly(hydroxymethacrylates), poly(hydroxypropylmethacrylamides),poly(hydroxypropyl methacrylates), poly(hydroxypropyloxazolines),poly(iminocarbonates), poly(lactic acids), poly(lactic-co-glycolicacids), poly(methacrylamides), poly(methacrylates),poly(methyloxazolines), poly(organophosphazenes), poly(ortho esters),poly(oxazolines), poly(propylene glycols), poly(siloxanes),poly(urethanes), poly(vinyl alcohols), poly(vinyl amines),poly(vinylmethylethers), poly(vinylpyrrolidones), silicones, celluloses,carbomethyl celluloses, hydroxypropyl methylcelluloses, chitins,chitosans, dextrans, dextrins, gelatins, hyaluronic acids andderivatives, functionalized hyaluronic acids, mannans, pectins,rhamnogalacturonans, starches, hydroxyalkyl starches, hydroxyethylstarches and other carbohydrate-based polymers, xylans, and copolymersthereof.

If the carrier —Z′ is a hydrogel, it is preferably a hydrogel comprisingPEG or hyaluronic acid. Most preferably such hydrogel comprises PEG. Inan equally preferred embodiment such hydrogel comprises hyaluronic acid.

Even more preferably, the carrier —Z′ is a hydrogel as described in WO2006/003014 A2, WO 2011/012715 A1 or WO 2014/056926 A1, which areherewith incorporated by reference in their entirety.

In another embodiment —Z′ is a polymer network formed through thephysical aggregation of polymer chains, which physical aggregation ispreferably caused by hydrogen bonds, crystallization, helix formation orcomplexation. In one embodiment such polymer network is a thermogellingpolymer.

In another embodiment the controlled-release CNP agonist is watersoluble.

In one embodiment the CNP agonist is a polypeptide or protein and thecontrolled-release CNP agonist is a fusion protein comprising suchpolypeptide or protein CNP agonist moiety fused to one or more furtherpolypeptide or protein moiety. Preferably, the CNP agonist is releasedfrom the fusion protein through enzymatic cleavage. Preferably, such atleast one or more further polypeptide or protein moieties are selectedfrom the group consisting of carboxyl-terminal peptide of the chorionicgonadotropin as described in US 2012/0035101 A1 which are herewithincorporated by reference; albumin; XTEN sequences as described in WO2011123813 A2 which are herewith incorporated by reference;proline/alanine random coil sequences as described in WO 2011/144756 A1which are herewith incorporated by reference; proline/alanine/serinerandom coil sequences as described in WO 2008/155134 A1 and WO2013/024049 A1 which are herewith incorporated by reference; and Fcfusion proteins.

In a preferred embodiment the controlled-release CNP agonist is a CNPagonist prodrug of formula (Ia) or (Ib)Z

L²-L¹-D)_(x)  (Ia)D

L²-L²-Z)_(y)  (Ib)

-   -   wherein        -   -D is a CNP agonist moiety;        -   -L- is a reversible prodrug linker moiety;        -   -L²- is a single chemical bond or a spacer moiety;        -   —Z is a water-soluble carrier moiety;        -   x is an integer selected from the group consisting of 1, 2,            3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16; and        -   y is an integer selected from the group consisting of 1, 2,            3, 4 and 5.

Preferably, x of formula (Ia) is an integer selected from the groupconsisting of 1, 2, 3, 4, 6 and 8. More preferably x of formula (Ia) isan integer selected from the group consisting of 1, 2, 4, and 6. Evenmore preferably x of formula (Ia) is an integer selected from the groupconsisting of 1, 4 and 6 and most preferably x of formula (Ia) is 1.

Preferably, y of formula (Ib) is an integer selected from the groupconsisting of 2, 3, 4 and 5, even more preferably an integer selectedfrom the group consisting of 2, 3 or 4 and most preferably an integerselected from the group consisting of 2 or 3.

In another preferred embodiment y of formula (Ib) is an integer selectedfrom the group consisting of 1, 2 or 3. In one preferred embodiment y offormula (Ib) is 1. In an equally preferred embodiment y of formula (Ib)is 2.

Preferably the controlled-release CNP agonist is a CNP agonist prodrugof formula (Ia) with x=1.

The moiety -L¹- is a reversible prodrug linker from which the drug, i.e.the CNP agonist, is released in its free form, i.e. -L- is a tracelessprodrug linker. Suitable prodrug linkers are known in the art, such asfor example the reversible prodrug linker moieties disclosed in WO2005/099768 A2, WO 2006/136586 A2, WO 2011/089216 A1 and WO 2013/024053A1, which are incorporated by reference herewith.

In another embodiment -L¹- is a reversible prodrug linker as describedin WO 2011/012722 A1, WO 2011/089214 A1, WO 2011/089215 A1, WO2013/024052 A1 and WO 2013/160340 A1 which are incorporated by referenceherewith.

The moiety -L¹- can be connected to -D through any type of linkage,provided that it is reversible. Preferably, -L¹- is connected to -Dthrough a linkage selected from the group consisting of amide, ester,carbamate, acetal, aminal, imine, oxime, hydrazone, disulfide andacylguanidine. Even more preferably -L- is connected to -D through alinkage selected from the group consisting of amide, ester, carbamateand acylguanidine. It is understood that these linkages may not per sebe reversible, but that neighboring groups comprised in -L¹- may renderthe linkage reversible.

In a preferred embodiment, the moiety -L¹- is connected to -D through anamide linkage.

A particularly preferred moiety -L¹- is disclosed in WO 2009/095479 A2.Accordingly, in one preferred embodiment the moiety -L¹- is of formula(II):

-   -   wherein the dashed line indicates the attachment to a nitrogen        of -D which is a CNP agonist moiety by forming an amide bond;    -   —X— is —C(R⁴R^(4a))—; —N(R⁴)—; —O—; —C(R⁴R^(4a))—C(R⁵R^(5a))—;        —C(R⁵R^(5a))—C(R⁴R^(4a))—; —C(R⁴R^(4a))—N(R⁶)—;        —N(R⁶)—C(R⁴R^(4a))—; —C(R⁴R^(4a))—O—; —O—C(R⁴R^(4a))—; or        —C(R⁷R^(7a))—;    -   X¹ is C; or S(O);    -   —X²— is —C(R⁸R^(8a))—; or —C(R⁸R^(8a))—C(R⁹R^(9a))—;    -   ═X³ is ═O; ═S; or ═N—CN;    -   —R¹, —R^(1a), —R², —R^(2a), —R⁴, —R^(4a), —R⁵, —R^(5a), —R⁶,        —R⁸, —R^(8a), —R⁹, R^(9a) are independently selected from the        group consisting of —H; and C₁₋₆ alkyl;    -   —R³, —R^(3a) are independently selected from the group        consisting of —H; and C₁₋₆ alkyl, provided that in case one of        —R³, —R^(3a) or both are other than —H they are connected to N        to which they are attached through an SP³-hybridized carbon        atom;    -   R⁷ is —N(R¹⁰R^(10a)); or —NRO—(C═O)—R¹¹;    -   —R^(7a), —R¹⁰, —R^(10a), —R¹¹ are independently of each other        —H; or C₁₋₆ alkyl;    -   optionally, one or more of the pairs —R^(1a)/—R^(4a),        —R^(1a)/—R^(5a), —R^(1a)/—R^(7a), —R^(4a)/—R^(5a),        —R^(8a)/—R^(9a) form a chemical bond;    -   optionally, one or more of the pairs —R¹/—R^(1a), —R²/—R^(2a),        —R⁴/—R^(4a), —R⁵/—R^(5a), —R/—R^(8a), —R⁹/—R^(9a) are joined        together with the atom to which they are attached to form a        C₃₋₁₀ cycloalkyl; or 3- to 10-membered heterocyclyl;    -   optionally, one or more of the pairs —R¹/—R⁴, —R¹/—R⁵, —R¹/—R⁶,        —R¹/—R^(7a), —R⁴/—R⁵, —R⁴/—R⁶, —R⁸/—R⁹, —R²/—R³ are joined        together with the atoms to which they are attached to form a        ring A;    -   optionally, R³/R^(3a) are joined together with the nitrogen atom        to which they are attached to form a 3- to 10-membered        heterocycle;    -   A is selected from the group consisting of phenyl; naphthyl;        indenyl; indanyl; tetralinyl; C₃₋₁₀ cycloalkyl; 3- to        10-membered heterocyclyl; and 8- to 11-membered heterobicyclyl;        and    -   wherein -L¹- is substituted with -L²-Z or -L²-Z′ and wherein        -L¹- is optionally further substituted, provided that the        hydrogen marked with the asterisk in formula (II) is not        replaced by -L²-Z or -L²-Z′ or a substituent;        -   wherein        -   L²- is a single chemical bond or a spacer;        -   —Z is a water-soluble carrier; and        -   Z′ is a water-insoluble carrier.

Preferably -L¹- of formula (II) is substituted with one moiety -L²-Z or-L²-Z′.

In one embodiment -L¹- of formula (II) is not further substituted.

It is understood that if —R³/—R^(3a) of formula (II) are joined togetherwith the nitrogen atom to which they are attached to form a 3- to10-membered heterocycle, only such 3- to 10-membered heterocycles may beformed in which the atoms directly attached to the nitrogen areSP³-hybridized carbon atoms. In other words, such 3- to 10-memberedheterocycle formed by —R³/—R^(3a) together with the nitrogen atom towhich they are attached has the following structure:

-   -   wherein    -   the dashed line indicates attachment to the rest of -L¹-;    -   the ring comprises 3 to 10 atoms comprising at least one        nitrogen; and    -   R^(#) and R## represent an SP³-hydridized carbon atom.

It is also understood that the 3- to 10-membered heterocycle may befurther substituted.

Exemplary embodiments of suitable 3- to 10-membered heterocycles formedby —R³/—R^(3a) of formula (II) together with the nitrogen atom to whichthey are attached are the following:

-   -   wherein    -   dashed lines indicate attachment to the rest of the molecule;        and    -   —R is selected from the group consisting of —H and C₁₋₆ alkyl.

-L¹- of formula (II) may optionally be further substituted. In general,any substituent may be used as far as the cleavage principle is notaffected, i.e. the hydrogen marked with the asterisk in formula (II) isnot replaced and the nitrogen of the moiety

of formula (II) remains part of a primary, secondary or tertiary amine,i.e. —R³ and —R^(3a) are independently of each other —H or are connectedto —N< through an SP³-hybridized carbon atom.

In one embodiment —R¹ or —R^(1a) of formula (II) is substituted with-L²-Z or -L²-Z′. In another embodiment —R² or —R^(2a) Of formula (II) issubstituted with -L²-Z or -L²-Z′. In another embodiment —R³ or —R^(3a)of formula (II) is substituted with -L²-Z or -L²-Z′. In anotherembodiment —R⁴ of formula (II) is substituted with -L²-Z or -L²-Z′. Inanother embodiment —R⁵ or —R^(5a) of formula (II) is substituted with-L²-Z or -L²-Z′. In another embodiment —R⁶ of formula (II) issubstituted with -L²-Z or -L²-Z′. In another embodiment —R⁷ or —R^(7a)of formula (II) is substituted with -L²-Z or -L²-Z′. In anotherembodiment —R⁸ or —R^(a) of formula (II) is substituted with -L²-Z or-L²-Z′. In another embodiment —R⁹ or —R^(9a) of formula (II) issubstituted with -L²-Z or -L²-Z′.

Most preferably —R⁴ of formula (II) is substituted with -L²-Z or -L²-Z′.

Preferably, —X— of formula (II) is —C(R⁴R^(4a))— or —N(R⁴)—. Mostpreferably, —X— of formula (II) is —C(R⁴R^(4a))—.

Preferably, X¹ of formula (II) is C.

Preferably, ═X³ of formula (II) is ═O.

Preferably, —X²— of formula (II) is —C(R⁸R^(8a))—.

Preferably —R⁸ and —R^(8a) of formula (II) are independently selectedfrom the group consisting of —H, methyl and ethyl. More preferably atleast one of —R⁸ and —R^(8a) of formula (II) is —H. Even more preferablyboth —R⁸ and —R^(8a) of formula (II) are —H.

Preferably, —R¹ and —R^(1a) of formula (II) are independently selectedfrom the group consisting of —H, methyl and ethyl. More preferably, atleast one of —R¹ and —R^(1a) of formula (II) is —H. Even more preferablyboth —R¹ and —R^(1a) of formula (II) are —H.

Preferably, —R² and —R^(2a) of formula (II) are independently selectedfrom the group consisting of —H, methyl and ethyl. More preferably, atleast one of —R² and —R^(2a) of formula (II) is —H. Even more preferablyboth —R² and —R^(2a) of formula (II) are H.

Preferably, —R³ and —R^(3a) of formula (II) are independently selectedfrom the group consisting of —H, methyl, ethyl, propyl and butyl. Evenmore preferably at least one of —R³ and —R^(3a) of formula (II) ismethyl. In an equally preferred embodiment —R³ and —R^(3a) of formula(II) are both —H. In another equally preferred embodiment —R³ and—R^(3a) of formula (II) are both methyl.

Preferably, —R³ of formula (II) is —H and —R^(3a) of formula (II) ismethyl.

Preferably, —R⁴ and —R^(4a) of formula (II) are independently selectedfrom the group consisting of —H, methyl and ethyl. More preferably, atleast one of —R⁴ and —R^(4a) of formula (II) is —H. Even more preferablyboth —R⁴ and —R^(4a) of formula (II) are —H.

Preferably the moiety -L¹- is of formula (IIa):

-   -   wherein the dashed line indicates the attachment to a nitrogen        of -D which is a CNP agonist moiety by forming an amide bond;    -   —R¹, —R^(1a), —R², —R^(2a), —R³, —R^(3a), —R⁴, —R^(4a) and —X²—        are used as defined in formula (II); and    -   wherein -L¹- is substituted with -L²-Z or -L²-Z′ and wherein        -L¹- is optionally further substituted, provided that the        hydrogen marked with the asterisk in formula (IIa) is not        replaced by -L²-Z or -L²-Z′ or a substituent.

Preferably -L¹- of formula (IIa) is substituted with one moiety -L²-Z or-L²-Z′.

Preferably the moiety -L¹- of formula (IIa) is not further substituted.

Preferably, —R¹ and —R^(1a) of formula (IIa) are independently selectedfrom the group consisting of —H, methyl and ethyl. More preferably, atleast one of —R¹ and —R^(1a) of formula (IIa) is —H. Even morepreferably both —R¹ and —R^(1a) of formula (IIa) are —H.

Preferably, —R⁴ and —R^(4a) of formula (IIa) are independently selectedfrom the group consisting of —H, methyl and ethyl. More preferably, atleast one of —R⁴ and —R^(4a) of formula (IIa) is —H. Even morepreferably both —R⁴ and —R^(4a) of formula (IIa) are —H.

Preferably, —X²— of formula (IIa) is —C(R⁸R^(8a))—.

Preferably —R⁸ and —R^(8a) of formula (IIa) are independently selectedfrom the group consisting of —H, methyl and ethyl. More preferably atleast one of —R⁸ and —R^(8a) of formula (IIa) is —H. Even morepreferably both —R⁸ and —R^(8a) of formula (IIa) are —H.

Preferably, —R² and —R^(2a) of formula (IIa) are independently selectedfrom the group consisting of —H, methyl and ethyl. More preferably, atleast one of —R² and —R^(2a) of formula (IIa) is —H. Even morepreferably both —R² and —R^(2a) of formula (IIa) are H.

Preferably, —R³ and —R^(3a) of formula (IIa) are independently selectedfrom the group consisting of —H, methyl, ethyl, propyl and butyl. Evenmore preferably at least one of —R³ and —R^(3a) of formula (IIa) ismethyl. In an equally preferred embodiment —R³ and —R^(3a) of formula(IIa) are both —H. In another equally preferred embodiment —R³ and—R^(3a) of formula (IIa) are both methyl.

Preferably, —R³ of formula (IIa) is —H and —R^(3a) of formula (IIa) ismethyl.

Preferably the moiety -L¹- is of formula (IIb):

-   -   wherein the dashed line indicates the attachment to a nitrogen        of -D which is a CNP agonist moiety by forming an amide bond;    -   —R², —R^(2a), —R³, —R^(3a) and —X²— are used as defined in        formula (II); and    -   wherein -L¹- is substituted with -L²-Z or -L²-Z′ and wherein        -L¹- is optionally further substituted, provided that the        hydrogen marked with the asterisk in formula (IIb) is not        replaced by -L²-Z or -L²-Z′ or a substituent.

Preferably -L¹- of formula (IIb) is substituted with one moiety -L²-Z or-L²-Z′.

Preferably the moiety -L¹- of formula (IIb) is not further substituted.

Preferably, —X²— of formula (IIb) is —C(R⁸R^(8a))—.

Preferably —R⁸ and —R^(8a) of formula (IIb) are independently selectedfrom the group consisting of —H, methyl and ethyl. More preferably atleast one of —R⁸ and —R^(8a) of formula (IIb) is —H.

Even more preferably both —R⁸ and —R^(8a) of formula (IIb) are —H.

Preferably, —R² and —R^(2a) of formula (IIb) are independently selectedfrom the group consisting of —H, methyl and ethyl. More preferably, atleast one of —R² and —R^(2a) of formula (IIb) is —H.

Even more preferably both —R² and —R^(2a) of formula (IIb) are H.

Preferably, —R³ and —R^(3a) of formula (IIb) are independently selectedfrom the group consisting of —H, methyl, ethyl, propyl and butyl. Evenmore preferably at least one of —R³ and —R^(3a) of formula (IIb) ismethyl. In an equally preferred embodiment —R³ and —R^(3a) of formula(IIb) are both —H. In another equally preferred embodiment —R³ and—R^(3a) of formula (IIb) are both methyl.

Most preferably, —R³ of formula (IIb) is —H and —R^(3a) of formula (IIb)is methyl.

Even more preferably the moiety -L¹- is of formula (IIb′):

-   -   wherein    -   wherein the dashed line indicates the attachment to a nitrogen        of D which is a CNP agonist moiety by forming an amide bond;    -   the dashed line marked with the asterisk indicates attachment to        -L²-;    -   —R², —R^(2a), —R³, —R^(3a) and —X²— are used as defined in        formula (II); and    -   wherein -L¹- is optionally further substituted, provided that        the hydrogen marked with the asterisk in formula (IIb′) is not        replaced by a substituent.

Preferably the moiety -L¹- of formula (IIb′) is not further substituted.

Preferably, —X²— of formula (IIb′) is —C(R⁸R^(8a))—.

Preferably —R⁸ and —R^(8a) of formula (IIb′) are independently selectedfrom the group consisting of —H, methyl and ethyl. More preferably atleast one of —R⁸ and —R^(8a) of formula (IIb′) is —H. Even morepreferably both —R⁸ and —R^(8a) of formula (IIb′) are —H.

Preferably, —R² and —R^(2a) of formula (IIb′) are independently selectedfrom the group consisting of —H, methyl and ethyl. More preferably, atleast one of —R² and —R^(2a) of formula (IIb′) is —H. Even morepreferably both —R² and —R^(2a) of formula (IIb′) are H.

Preferably, —R³ and —R^(3a) of formula (IIb′) are independently selectedfrom the group consisting of —H, methyl, ethyl, propyl and butyl. Evenmore preferably at least one of —R³ and —R^(3a) of formula (IIb′) ismethyl. In an equally preferred embodiment —R³ and —R^(3a) of formula(IIb′) are both —H. In another equally preferred embodiment —R³ and—R^(3a) of formula (IIb′) are both methyl.

Most preferably, —R³ of formula (IIb′) is —H and —R^(3a) of formula(IIb′) is methyl.

Preferably the moiety -L¹- is of formula (IIc):

-   -   wherein the dashed line indicates the attachment to a nitrogen        of -D which is a CNP agonist moiety by forming an amide bond;        and    -   wherein -L¹- is substituted with -L²-Z or -L²-Z′ and wherein        -L¹- is optionally further substituted, provided that the        hydrogen marked with the asterisk in formula (IIc) is not        replaced by -L²-Z or -L²-Z′ or a substituent.

Preferably -L¹- of formula (IIc) is substituted with one moiety -L²-Z or-L²-Z′.

Preferably the moiety -L¹- of formula (IIc) is not further substituted.

In another preferred embodiment the moiety -L¹- is of formula (IIc-a):

-   -   wherein the dashed line indicates the attachment to a nitrogen        of -D which is a CNP agonist moiety by forming an amide bond;        and    -   wherein -L¹- is substituted with -L²-Z or -L²-Z′ and wherein        -L¹- is optionally further substituted, provided that the        hydrogen marked with the asterisk in formula (IIc-a) is not        replaced by -L²-Z or -L²-Z′ or a substituent.

Preferably -L¹- of formula (IIc-a) is substituted with one moiety -L²-Zor -L²-Z′.

Preferably the moiety -L¹- of formula (IIc-a) is not furthersubstituted.

In another preferred embodiment the moiety -L¹- is of formula (IIc-b):

-   -   wherein the dashed line indicates the attachment to a nitrogen        of -D which is a CNP agonist moiety by forming an amide bond;        and    -   wherein -L¹- is substituted with -L²-Z or -L²-Z′ and wherein        -L¹- is optionally further substituted, provided that the        hydrogen marked with the asterisk in formula (IIc-b) is not        replaced by -L²-Z or -L²-Z′ or a substituent.

Preferably -L- of formula (IIc-b) is substituted with one moiety -L²-Zor -L²-Z′.

Preferably the moiety -L¹- of formula (IIc-b) is not furthersubstituted.

Even more preferably the moiety -L- is selected from the groupconsisting of formula (IIc-i), (Hc-ii), (IIce-iii), (IIe-iv) and(IIce-v):

-   -   wherein    -   the unmarked dashed line indicates the attachment to a nitrogen        of -D which is a CNP agonist moiety by forming an amide bond;    -   the dashed line marked with the asterisk indicates attachment to        -L²-Z or -L²-Z′; and    -   -L¹- is optionally further substituted, provided that the        hydrogen marked with the asterisk in formula (IIc-i), (IIc-ii),        (IIc-iii), (IIc-iv) and (IIc-v) is not replaced by a        substituent.

Preferably, the moiety -L¹- of formula (IIc-i), (IIc-ii), (IIc-iii),(IIc-iv) and (IIc-v) is not further substituted.

In a particularly preferred embodiment the moiety -L¹- is of formula(IIc-ii)

-   -   wherein    -   the unmarked dashed line indicates the attachment to a nitrogen        of -D which is a CNP agonist moiety by forming an amide bond;        and    -   the dashed line marked with the asterisk indicates attachment to        -L²-Z or -L²-Z′.

Preferably -L¹- of formula (IIc-ii) is substituted with one moiety -L²-Zor -L²-Z′.

In an equally preferred embodiment the moiety -L¹- is selected from thegroup consisting of formula (IIc-i′), (IIc-ii′), (IIc-iii′), (IIc-iv′)and (IIc-v′):

-   -   wherein    -   the unmarked dashed line indicates the attachment to a nitrogen        of -D which is a CNP agonist moiety by forming an amide bond;    -   the dashed line marked with the asterisk indicates attachment to        -L²-Z or -L²-Z′; and    -   L- is optionally further substituted, provided that the hydrogen        marked with the asterisk in formula (IIc-i′), (IIc-ii′),        (IIc-iii′), (IIc-iv′) and (IIc-v′) is not replaced by a        substituent.

Preferably, the moiety -L¹- of formula (IIc-i′), (IIc-ii′), (IIc-iii′),(IIc-iv′) and (IIc-v′) is not further substituted.

In another particularly preferred embodiment the moiety -L¹- is offormula (IIc-ii′)

-   -   wherein    -   the unmarked dashed line indicates the attachment to a nitrogen        of -D which is a CNP agonist moiety by forming an amide bond;        and    -   the dashed line marked with the asterisk indicates attachment to        -L²-Z or -L²-Z′.

Preferably -L¹- of formula (IIc-ii′) is substituted with one moiety-L²-Z or -L²-Z′.

In an equally preferred embodiment the moiety -L- is selected from thegroup consisting of formula (IIc-i″), (IIc-ii″), (IIc-iii″) and(IIc-iv″):

-   -   wherein    -   the unmarked dashed line indicates the attachment to a nitrogen        of -D which is a CNP agonist moiety by forming an amide bond;    -   the dashed line marked with the asterisk indicates attachment to        -L²-Z or -L²-Z′; and    -   L¹- is optionally further substituted, provided that the        hydrogen marked with the asterisk in formula (IIc-i″),        (IIc-ii″), (IIc-iii″) and (IIc-iv″) is not replaced by a        substituent.

Preferably, the moiety -L¹- of formula (IIc-i″), (IIc-ii″), (IIc-iii″)and (IIc-iv″) is not further substituted.

In another particularly preferred embodiment the moiety -L¹- is offormula (IIc-ii″)

-   -   wherein    -   the unmarked dashed line indicates the attachment to a nitrogen        of -D which is a CNP agonist moiety by forming an amide bond;        and    -   the dashed line marked with the asterisk indicates attachment to        -L²-Z or -L²-Z′.

Preferably -L¹- of formula (IIc-ii″) is substituted with one moiety-L²-Z or -L²-Z′.

The optional further substituents of -L¹- of formula (II), (IIa), (IIb),(IIb′), (IIc), (IIc-a), (IIc-b), (IIc-i), (IIc-ii), (IIc-iii), (IIc-iv),(IIc-v), (IIc-i′), (IIc-ii′), (IIc-iii′), (IIc-iv′), (IIc-v′), (IIc-i″),(IIc-ii″), (IIc-iii″), (IIc-iv″) are preferably as described above.

Another preferred moiety -L¹- is disclosed in WO2016/020373A1.Accordingly, in another preferred embodiment the moiety -L¹- is offormula (III):

-   -   wherein    -   the dashed line indicates attachment to a primary or secondary        amine or hydroxyl of -D which is a CNP moiety by forming an        amide or ester linkage, respectively;    -   —R¹, —R^(1a), —R², —R^(2a), —R³ and —R^(3a) are independently of        each other selected from the group consisting of —H,        —C(R⁸R^(8a)R^(8b)), —C(═O)R, —C═N, —C(═NR⁸)R^(8a),        —CR⁸(═C^(8a)R^(8b)), —C≡CR⁸ and -T;    -   —R⁴, —R⁵ and —R^(5a) are independently of each other selected        from the group consisting of —H, —C(R⁹R^(9a)R^(9b)) and -T;    -   a1 and a2 are independently of each other 0 or 1;    -   each —R⁶, —R^(6a), —R⁷, —R^(7a), —R⁸, —R^(8a), —R^(8b), —R⁹,        —R^(9a), —R^(9b) are independently of each other selected from        the group consisting of —H, halogen, —CN, —COOR¹⁰, —OR¹⁰,        —C(O)R¹⁰, —C(O)N(R¹⁰R^(10a)), —S(O)₂N(R¹⁰R^(10a)),        —S(O)N(R₁₀R^(10a)), —S(O)₂R¹⁰, —S(O)R¹⁰,        —N(R¹⁰)S(O)₂N(R^(10a)R^(10b)), —SR¹⁰, —N(R₁₀R^(10a)), —NO₂,        —OC(O)R¹⁰, —N(R¹⁰)C(O)R^(10a), —N(R¹⁰)S(O)₂R^(10a),        —N(R¹⁰)S(O)R^(10a), —N(R¹⁰)C(O)OR^(10a),        —N(R¹⁰)C(O)N(R^(10a)R^(10b)), —OC(O)N(R¹⁰R^(10a)), -T, C₁₋₂₀        alkyl, C₂₋₂₀ alkenyl, and C₂₋₂₀ alkynyl; wherein -T, C₁₋₂₀        alkyl, C₂₋₂₀ alkenyl, and C₂₋₂₀ alkynyl are optionally        substituted with one or more —R¹¹, which are the same or        different and wherein C₁₋₂₀ alkyl, C₂₋₂₀ alkenyl, and C₂₋₂₀        alkynyl are optionally interrupted by one or more groups        selected from the group consisting of -T-, —C(O)O—, —O—, —C(O)—,        —C(O)N(R¹²)—, —S(O)₂N(R¹²)—, —S(O)N(R¹²)—, —S(O)₂—, —S(O)—,        —N(R¹²)S(O)₂N(R^(12a))—, —S—, —N(R¹²)—, —OC(OR¹²)(R^(12a))—,        —N(R¹²)C(O)N(R^(12a))—, and —OC(O)N(R¹²)—;    -   each —R¹⁰, —R^(10a), —R^(10b) is independently selected from the        group consisting of —H, -T, C₁₋₂₀ alkyl, C₂₋₂₀ alkenyl, and        C₂₋₂₀ alkynyl; wherein -T, C₁₋₂₀ alkyl, C₂₋₂₀ alkenyl, and C₂₋₂₀        alkynyl are optionally substituted with one or more —R¹¹, which        are the same or different and wherein C₁₋₂₀ alkyl, C₂₋₂₀        alkenyl, and C₂₋₂₀ alkynyl are optionally interrupted by one or        more groups selected from the group consisting of -T-, —C(O)O—,        —O—, —C(O)—, —C(O)N(R¹²)—, —S(O)₂N(R¹²)—, —S(O)N(R¹²)—, —S(O)₂—,        —S(O)—, —N(R¹²)S(O)₂N(R^(12a))—, —S—, —N(R¹²)—,        —OC(OR¹²)(R^(12a))—, —N(R¹²)C(O)N(R^(12a))—, and —OC(O)N(R¹²)—;    -   each T is independently of each other selected from the group        consisting of phenyl, naphthyl, indenyl, indanyl, tetralinyl,        C₃₋₁₀ cycloalkyl, 3- to 10-membered heterocyclyl, and 8- to        11-membered heterobicyclyl; wherein each T is independently        optionally substituted with one or more —R¹¹, which are the same        or different;    -   each —R¹¹ is independently of each other selected from halogen,        —CN, oxo (═O), —COOR¹³, —OR¹³, —C(O)R¹³, —C(O)N(R¹³R^(13a)),        —S(O)₂N(R¹³R^(13a)), —S(O)N(R¹³R^(13a)), —S(O)₂R¹³, —S(O)R¹³,        —N(R¹³)S(O)₂N(R^(13a)R^(13b)), —SR¹³, —N(R¹³R^(13a)), —NO₂,        —OC(O)R¹³, —N(R¹³)C(O)R^(13a), —N(R¹³)S(O)₂R^(13a),        —N(R¹³)S(O)R^(13a), —N(R¹³)C(O)OR^(13a),        —N(R¹³)C(O)N(R^(13a)R^(13b)), —OC(O)N(R¹³R^(13a)), and C₁₋₆        alkyl; wherein C₁₋₆ alkyl is optionally substituted with one or        more halogen, which are the same or different;    -   each —R¹², —R^(12a), —R¹³, —R^(13a), R^(13b) is independently        selected from the group consisting of —H, and C₁₋₆ alkyl;        wherein C₁₋₆ alkyl is optionally substituted with one or more        halogen, which are the same or different;    -   optionally, one or more of the pairs —R¹/—R^(1a), —R²/—R^(2a),        —R³/—R^(3a), —R⁶/—R^(6a), —R⁷/—R^(7a) are joined together with        the atom to which they are attached to form a C₃₋₁₀ cycloalkyl        or a 3- to 10-membered heterocyclyl;    -   optionally, one or more of the pairs —R¹/—R², —R¹/—R³, —R¹/—R⁴,        —R¹/—R⁵, —R¹/—R⁶, —R¹/—R, —R²/—R³, —R²/—R⁴, —R²/—R⁵, —R²/—R⁶,        —R²/—R⁷, —R³/—R⁴, —R³/—R⁵, —R³/—R⁶, —R³/—R⁷, —R⁴/—R⁵, —R⁴/—R⁶,        —R⁴/—R⁷, —R⁵/—R⁶, —R⁵/—R⁷, —R⁶/—R⁷ are joint together with the        atoms to which they are attached to form a ring A;    -   A is selected from the group consisting of phenyl; naphthyl;        indenyl; indanyl; tetralinyl; C₃₋₁₀ cycloalkyl; 3- to        10-membered heterocyclyl; and 8- to 11-membered heterobicyclyl;    -   wherein -L¹- is substituted with -L²-Z or -L²-Z′ and wherein        -L¹- is optionally further substituted;        -   wherein        -   L²- is a single chemical bond or a spacer;        -   —Z is a water-soluble carrier; and        -   —Z′ is a water-insoluble carrier.

The optional further substituents of -L¹- of formula (III) arepreferably as described above.

Preferably -L¹- of formula (III) is substituted with one moiety -L²-Z or-L²-Z′.

In one embodiment -L¹- of formula (III) is not further substituted.

Additional preferred embodiments for -L¹- are disclosed in EP1536334B1,WO2009/009712A1, WO2008/034122A1, WO2009/143412A2, WO2011/082368A2, andU.S. Pat. No. 8,618,124B2, which are herewith incorporated by referencein their entirety.

Additional preferred embodiments for -L- are disclosed in U.S. Pat. No.8,946,405B2 and U.S. Pat. No. 8,754,190B2, which are herewithincorporated by reference in their entirety. Accordingly, a preferredmoiety -L¹- is of formula (IV):

-   -   wherein    -   the dashed line indicates attachment to -D which is a CNP        agonist moiety and wherein attachment is through a functional        group of -D selected from the group consisting of —OH, —SH and        —NH₂;    -   m is 0 or 1;    -   at least one or both of —R¹ and —R² is/are independently of each        other selected from the group consisting of —CN, —NO₂,        optionally substituted aryl, optionally substituted heteroaryl,        optionally substituted alkenyl, optionally substituted alkynyl,        —C(O)R³, —S(O)R³, —S(O)₂R³, and —SR⁴,    -   one and only one of —R¹ and —R² is selected from the group        consisting of —H, optionally substituted alkyl, optionally        substituted arylalkyl, and optionally substituted        heteroarylalkyl;    -   R³ is selected from the group consisting of —H, optionally        substituted alkyl, optionally substituted aryl, optionally        substituted arylalkyl, optionally substituted heteroaryl,        optionally substituted heteroarylalkyl, —OR⁹ and —N(R⁹)₂;    -   —R⁴ is selected from the group consisting of optionally        substituted alkyl, optionally substituted aryl, optionally        substituted arylalkyl, optionally substituted heteroaryl, and        optionally substituted heteroarylalkyl;    -   each —R⁵ is independently selected from the group consisting of        —H, optionally substituted alkyl, optionally substituted        alkenylalkyl, optionally substituted alkynylalkyl, optionally        substituted aryl, optionally substituted arylalkyl, optionally        substituted heteroaryl and optionally substituted        heteroarylalkyl;    -   R⁹ is selected from the group consisting of —H and optionally        substituted alkyl;    -   —Y— is absent and —X— is —O— or —S—; or    -   —Y— is —N(Q)CH₂— and —X— is —O—;    -   Q is selected from the group consisting of optionally        substituted alkyl, optionally substituted aryl, optionally        substituted arylalkyl, optionally substituted heteroaryl and        optionally substituted heteroarylalkyl;    -   optionally, —R¹ and —R² may be joined to form a 3 to 8-membered        ring; and    -   optionally, both —R⁹ together with the nitrogen to which they        are attached form a heterocyclic ring;    -   wherein -L¹- is substituted with -L²-Z or -L²-Z′ and wherein        -L¹- is optionally further substituted;    -   wherein    -   L²- is a single chemical bond or a spacer;    -   —Z is a water-soluble carrier; and    -   —Z′ is a water-insoluble carrier.

The optional further substituents of -L¹- of formula (IV) are preferablyas described above.

Preferably -L¹- of formula (IV) is substituted with one moiety -L²-Z or-L²-Z′.

In one embodiment -L- of formula (IV) is not further substituted.

Only in the context of formula (IV) the terms used have the followingmeaning:

The term “alkyl” as used herein includes linear, branched or cyclicsaturated hydrocarbon groups of 1 to 8 carbons, or in some embodiments 1to 6 or 1 to 4 carbon atoms.

The term “alkoxy” includes alkyl groups bonded to oxygen, includingmethoxy, ethoxy, isopropoxy, cyclopropoxy, cyclobutoxy, and similar.

The term “alkenyl” includes non-aromatic unsaturated hydrocarbons withcarbon-carbon double bonds.

The term “alkynyl” includes non-aromatic unsaturated hydrocarbons withcarbon-carbon triple bonds.

The term “aryl” includes aromatic hydrocarbon groups of 6 to 18 carbons,preferably 6 to 10 carbons, including groups such as phenyl, naphthyl,and anthracenyl. The term “heteroaryl” includes aromatic ringscomprising 3 to 15 carbons containing at least one N, O or S atom,preferably 3 to 7 carbons containing at least one N, O or S atom,including groups such as pyrrolyl, pyridyl, pyrimidinyl, imidazolyl,oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, quinolyl, indolyl,indenyl, and similar.

In some instance, alkenyl, alkynyl, aryl or heteroaryl moieties may becoupled to the remainder of the molecule through an alkylene linkage.Under those circumstances, the substituent will be referred to asalkenylalkyl, alkynylalkyl, arylalkyl or heteroarylalkyl, indicatingthat an alkylene moiety is between the alkenyl, alkynyl, aryl orheteroaryl moiety and the molecule to which the alkenyl, alkynyl, arylor heteroaryl is coupled.

The term “halogen” includes bromo, fluoro, chloro and iodo.

The term “heterocyclic ring” refers to a 4 to 8 membered aromatic ornon-aromatic ring comprising 3 to 7 carbon atoms and at least one N, O,or S atom. Examples are piperidinyl, piperazinyl, tetrahydropyranyl,pyrrolidine, and tetrahydrofuranyl, as well as the exemplary groupsprovided for the term “heteroaryl” above.

When a ring system is optionally substituted, suitable substituents areselected from the group consisting of alkyl, alkenyl, alkynyl, or anadditional ring, each optionally further substituted.

Optional substituents on any group, including the above, include halo,nitro, cyano, —OR, —SR, —NR₂, —OCOR, —NRCOR, —COOR, —CONR₂, —SOR, —SO₂R,—SONR₂, —SO₂NR₂, wherein each R is independently alkyl, alkenyl,alkynyl, aryl or heteroaryl, or two R groups taken together with theatoms to which they are attached form a ring.

An additional preferred embodiment for -L¹- is disclosed inWO2013/036857A1, which is herewith incorporated by reference in itsentirety. Accordingly, a preferred moiety -L¹- is of formula (V):

-   -   wherein    -   the dashed line indicates attachment to -D which is a CNP        agonist moiety and wherein attachment is through an amine        functional group of -D;    -   —R¹ is selected from the group consisting of optionally        substituted C₁-C₆ linear, branched, or cyclic alkyl; optionally        substituted aryl; optionally substituted heteroaryl; alkoxy; and        —NR⁵ ₂;    -   —R² is selected from the group consisting of —H; optionally        substituted C₁-C₆ alkyl; optionally substituted aryl; and        optionally substituted heteroaryl;    -   R³ is selected from the group consisting of —H; optionally        substituted C₁-C₆ alkyl; optionally substituted aryl; and        optionally substituted heteroaryl;    -   —R⁴ is selected from the group consisting of —H; optionally        substituted C₁-C₆ alkyl; optionally substituted aryl; and        optionally substituted heteroaryl;    -   each —R⁵ is independently of each other selected from the group        consisting of —H; optionally substituted C₁-C₆ alkyl; optionally        substituted aryl; and optionally substituted heteroaryl; or when        taken together two —R⁵ can be cycloalkyl or cycloheteroalkyl;    -   wherein -L¹- is substituted with -L²-Z or -L²-Z′ and wherein        -L¹- is optionally further substituted;        -   wherein        -   -L²- is a single chemical bond or a spacer;        -   —Z is a water-soluble carrier; and        -   —Z′ is a water-insoluble carrier.

The optional further substituents of -L¹- of formula (V) are preferablyas described above.

Preferably -L¹- of formula (V) is substituted with one moiety -L²-Z or-L²-Z′.

In one embodiment -L¹- of formula (V) is not further substituted.

Only in the context of formula (V) the terms used have the followingmeaning:

“Alkyl”, “alkenyl”, and “alkynyl” include linear, branched or cyclichydrocarbon groups of 1-8 carbons or 1-6 carbons or 1-4 carbons whereinalkyl is a saturated hydrocarbon, alkenyl includes one or morecarbon-carbon double bonds and alkynyl includes one or morecarbon-carbon triple bonds. Unless otherwise specified these contain 1-6C.

“Aryl” includes aromatic hydrocarbon groups of 6-18 carbons, preferably6-10 carbons, including groups such as phenyl, naphthyl, and anthracene“Heteroaryl” includes aromatic rings comprising 3-15 carbons containingat least one N, O or S atom, preferably 3-7 carbons containing at leastone N, O or S atom, including groups such as pyrrolyl, pyridyl,pyrimidinyl, imidazolyl, oxazolyl, isoxazolyl, thiszolyl, isothiazolyl,quinolyl, indolyl, indenyl, and similar.

The term “substituted” means an alkyl, alkenyl, alkynyl, aryl, orheteroaryl group comprising one or more substituent groups in place ofone or more hydrogen atoms. Substituents may generally be selected fromhalogen including F, Cl, Br, and I; lower alkyl including linear,branched, and cyclic; lower haloalkyl including fluoroalkyl,chloroalkyl, bromoalkyl, and iodoalkyl; OH; lower alkoxy includinglinear, branched, and cyclic; SH; lower alkylthio including linear,branched and cyclic; amino, alkylamino, dialkylamino, silyl includingalkylsilyl, alkoxysilyl, and arylsilyl; nitro; cyano; carbonyl;carboxylic acid, carboxylic ester, carboxylic amide, aminocarbonyl;aminoacyl; carbamate; urea; thiocarbamate; thiourea; ketne; sulfone;sulfonamide; aryl including phenyl, naphthyl, and anthracenyl;heteroaryl including 5-member heteroaryls including as pyrrole,imidazole, furan, thiophene, oxazole, thiazole, isoxazole, isothiazole,thiadiazole, triazole, oxadiazole, and tetrazole, 6-member heteroarylsincluding pyridine, pyrimidine, pyrazine, and fused heteroarylsincluding benzofuran, benzothiophene, benzoxazole, benzimidazole,indole, benzothiazole, benzisoxazole, and benzisothiazole.

A further preferred embodiment for -L¹- is disclosed in U.S. Pat. No.7,585,837B2, which is herewith incorporated by reference in itsentirety. Accordingly, a preferred moiety -L¹- is of formula (VI):

-   -   wherein    -   the dashed line indicates attachment to -D which is a CNP        agonist moiety and wherein attachment is through an amine        functional group of -D;    -   R¹ and R² are independently selected from the group consisting        of hydrogen, alkyl, alkoxy, alkoxyalkyl, aryl, alkaryl, aralkyl,        halogen, nitro, —SO₃H, —SO₂NHR⁵, amino, ammonium, carboxyl,        PO₃H₂, and OPO₃H₂;    -   R³, R⁴, and R⁵ are independently selected from the group        consisting of hydrogen, alkyl, and aryl;    -   wherein -L¹- is substituted with -L²-Z or -L²-Z′ and wherein        -L¹- is optionally further substituted;        -   wherein        -   -L²- is a single chemical bond or a spacer;        -   —Z is a water-soluble carrier; and        -   —Z′ is a water-insoluble carrier.

Suitable substituents for formulas (VI) are alkyl (such as C₁₋₆ alkyl),alkenyl (such as C₂₋₆ alkenyl), alkynyl (such as C₂₋₆ alkynyl), aryl(such as phenyl), heteroalkyl, heteroalkenyl, heteroalkynyl, heteroaryl(such as aromatic 4 to 7 membered heterocycle) or halogen moieties.

Preferably -L- of formula (VI) is substituted with one moiety -L²-Z or-L²-Z′. In one embodiment -L- of formula (VI) is not furthersubstituted.

Only in the context of formula (VI) the terms used have the followingmeaning:

The terms “alkyl”, “alkoxy”, “alkoxyalkyl”, “aryl”, “alkaryl” and“aralkyl” mean alkyl radicals of 1-8, preferably 1-4 carbon atoms, e.g.methyl, ethyl, propyl, isopropyl and butyl, and aryl radicals of 6-10carbon atoms, e.g. phenyl and naphthyl. The term “halogen” includesbromo, fluoro, chloro and iodo.

A further preferred embodiment for -L- is disclosed in WO2002/089789A1,which is herewith incorporated by reference in its entirety.Accordingly, a preferred moiety -L¹- is of formula (VII):

-   -   wherein    -   the dashed line indicates attachment to -D which is a CNP        agonist moiety and wherein attachment is through an amine        functional group of -D;    -   L₁ is a bifunctional linking group,    -   Y₁ and Y₂ are independently O, S or NR⁷;    -   R², R³, R⁴, R⁵, R⁶ and R⁷ are independently selected from the        group consisting of hydrogen, C₁₋₆ alkyls, C₃₋₁₂ branched        alkyls, C₃₋₈ cycloalkyls, C₁₋₆ substituted alkyls, C₃₋₈        substituted cycloalkyls, aryls, substituted aryls, aralkyls,        C₁₋₆ heteroalkyls, substituted C₁₋₆ heteroalkyls, C₁₋₆ alkoxy,        phenoxy, and C₁₋₆ heteroalkoxy;    -   Ar is a moiety which when included in formula (VII) forms a        multisubstituted aromatic hydrocarbon or a multi-substituted        heterocyclic group;    -   X is a chemical bond or a moiety that is actively transported        into a target cell, a hydrophobic moiety, or a combination        thereof,    -   y is 0 or 1;    -   wherein -L- is substituted with -L²-Z or -L²-Z′ and wherein -L¹-        is optionally further substituted;        -   wherein        -   L²- is a single chemical bond or a spacer;        -   —Z is a water-soluble carrier; and        -   Z′ is a water-insoluble carrier.

The optional further substituents of -L¹- of formula (VII) arepreferably as described above.

Preferably -L¹- of formula (VII) is substituted with one moiety -L²-Z or-L²-Z′.

In one embodiment -L¹- of formula (VII) is not further substituted.

Only in the context of formula (VII) the terms used have the followingmeaning:

The term “alkyl” shall be understood to include, e.g. straight,branched, substituted C₁₋₁₂ alkyls, including alkoxy, C₃₋₈ cycloalkylsor substituted cycloalkyls, etc.

The term “substituted” shall be understood to include adding orreplacing one or more atoms contained within a functional group orcompounds with one or more different atoms.

Substituted alkyls include carboxyalkyls, aminoalkyls, dialkylaminos,hydroxyalkyls and mercaptoalkyls; substituted cycloalkyls includemoieties such as 4-chlorocyclohexyl; aryls include moieties such asnapthyl; substituted aryls include moieties such as 3-bromo-phenyl;aralkyls include moieties such as toluyl; heteroalkyls include moietiessuch as ethylthiophene; substituted heteroalkyls include moieties suchas 3-methoxythiophone; alkoxy includes moieties such as methoxy; andphenoxy includes moieties such as 3-nitrophenoxy. Halo-shall beunderstood to include fluoro, chloro, iodo and bromo.

In another preferred embodiment -L¹- comprises a substructure of formula(VIII)

-   -   wherein    -   the dashed line marked with the asterisk indicates attachment to        a nitrogen of -D which is a CNP agonist moiety by forming an        amide bond;    -   the unmarked dashed lines indicate attachment to the remainder        of -L¹-; and    -   wherein -L¹- is substituted with -L²-Z or -L²-Z′ and wherein        -L¹- is optionally further substituted;        -   wherein        -   -L²- is a single chemical bond or a spacer;        -   —Z is a water-soluble carrier; and        -   —Z′ is a water-insoluble carrier.

The optional further substituents of -L¹- of formula (VIII) arepreferably as described above.

Preferably -L¹- of formula (VIII) is substituted with one moiety -L²-Zor -L²-Z′.

In one embodiment -L¹- of formula (VIII) is not further substituted.

In another preferred embodiment -L- comprises a substructure of formula(IX)

-   -   wherein    -   the dashed line marked with the asterisk indicates attachment to        a nitrogen of -D which is a CNP agonist moiety by forming a        carbamate bond;    -   the unmarked dashed lines indicate attachment to the remainder        of -L¹-; and    -   wherein -L¹- is substituted with -L²-Z or -L²-Z′ and wherein        -L¹- is optionally further substituted;        -   wherein        -   -L²- is a single chemical bond or a spacer;        -   —Z is a water-soluble carrier; and        -   —Z′ is a water-insoluble carrier.

The optional further substituents of -L¹- of formula (IX) are preferablyas described above.

Preferably -L¹- of formula (IX) is substituted with one moiety -L²-Z or-L²-Z′.

In one embodiment -L¹- of formula (IX) is not further substituted.

Preferably -D of formula (Ia), (Ib), (II), (IIa), (IIb), (IIb′), (IIc),(IIc-a), (IIc-b), (IIc-i), (IIc-ii), (IIc-iii), (IIc-iv), (IIc-v),(IIc-i′), (IIc-ii′), (IIc-iii′), (IIc-iv′), (IIc-v′), (IIc-i″),(IIc-ii″), (IIc-iii″), (IIc-iv″), (III), (IV), (V), (VI), (VII), (VIII)and (IX) is a CNP moiety. The moiety -D may be connected to -L¹- throughany functional group of D-H and is preferably connected to -L¹- throughan amine functional group of D-H. This may be the N-terminal aminefunctional group or an amine functional group provided by a lysine sidechain, i.e. by the lysines at position 9, 11, 15, 16, 20 and 26, if theCNP has the sequence of SEQ ID NO:24.

It was surprisingly found that attachment of -L¹- to the ring of a CNPmoiety significantly reduces the CNP prodrug's affinity to NPR-Bcompared to attachment at the N-terminus or to the non-ring part of CNP,which reduced affinity to NPR-B in turn reduces the risk ofcardiovascular side effects, such as hypotension.

Accordingly, -L¹- is preferably conjugated to the side chain of an aminoacid residue of said ring moiety of -D or to the backbone of said ringmoiety of -D. Even more preferably, -L¹- is covalently and reversiblyconjugated to the side chain of an amino acid residue of said ringmoiety of -D. If -D is a CNP moiety with the sequence of SEQ ID NO:24,-L¹- is preferably conjugated to the amine functional group provided bythe lysine at position 26 of the corresponding drug D-H.

The moiety -L²- can be attached to -L¹- by replacing any —H present,except where explicitly excluded.

The moiety -L²- is a chemical bond or a spacer moiety.

In one embodiment -L²- is a chemical bond.

In another embodiment -L²- is a spacer moiety.

When -L²- is other than a single chemical bond, -L²- is preferablyselected from the group consisting of -T-, —C(O)O—, —O—, —C(O)—,—C(O)N(R^(y1))—, —S(O)₂N(R′)—, —S(O)N(R^(y1))—, —S(O)₂—, —S(O)—,—N(R^(y1))S(O)₂N(R^(y1a))—, —S—, —N(R^(y1))—, —OC(OR^(y1))(R^(y1a))—,—N(R^(y1))C(O)N(R^(y1a))—, —OC(O)N(R^(y1))—, C₁₋₅₀ alkyl, C₂₋₅₀ alkenyl,and C₂₋₅₀ alkynyl; wherein -T-, C₁₋₅₀ alkyl, C₂₋₅₀ alkenyl, and C₂₋₅₀alkynyl are optionally substituted with one or more —R^(y2), which arethe same or different and wherein C₁₋₅₀ alkyl, C₂₋₅₀ alkenyl, and C₂₋₅₀alkynyl are optionally interrupted by one or more groups selected fromthe group consisting of -T-, —C(O)O—, —O—, —C(O)—, —C(O)N(R^(y3))—,—S(O)₂N(R^(y3))—, —S(O)N(R^(y3))—, —S(O)₂—, —S(O)—,—N(R^(y3))S(O)₂N(R^(y3a))—, —S—, —N(R^(y3))—, —OC(OR^(y3))(R^(y3a))—,—N(R^(y3))C(O)N(R^(y3a))—, and —OC(O)N(R³)—;

—R^(y1) and —R^(y1a) are independently of each other selected from thegroup consisting of —H, -T, C₁₋₅₀ alkyl, C₂₋₅₀ alkenyl, and C₂₋₅₀alkynyl; wherein -T, C₁₋₅₀ alkyl, C₂₋₅₀ alkenyl, and C₂₋₅₀ alkynyl areoptionally substituted with one or more —R^(y2), which are the same ordifferent, and wherein C₁₋₅₀ alkyl, C₂₋₅₀ alkenyl, and C₂₋₅₀ alkynyl areoptionally interrupted by one or more groups selected from the groupconsisting of -T-, —C(O)O—, —O—, —C(O)—, —C(O)N(R^(y4))—,—S(O)₂N(R^(y4))—, —S(O)N(R^(y4))—, —S(O)₂—, —S(O)—,—N(R^(y4))S(O)₂N(R^(y4a))—, —S—, —N(R^(y4))—, —OC(OR^(y4))(R^(y4a))—,—N(R^(y4))C(O)N(R^(y4a))—, and —OC(O)N(R^(y4))—;

each T is independently selected from the group consisting of phenyl,naphthyl, indenyl, indanyl, tetralinyl, C₃₋₁₀ cycloalkyl, 3- to10-membered heterocyclyl, 8- to 11-membered heterobicyclyl, 8- to30-membered carbopolycyclyl, and 8- to 30-membered heteropolycyclyl;wherein each T is independently optionally substituted with one or more—R^(y2), which are the same or different;

each —R^(y2) is independently selected from the group consisting ofhalogen, —CN, oxo (═O), —COOR^(y5), —OR^(y5), —C(O)R^(y5),—C(O)N(R^(y5)R^(y5a)), —S(O)₂N(R^(y5)R^(y5a)), —S(O)N(R^(y5)R^(y5a)),—S(O)₂R^(y5), —S(O)R^(y5), —N(R⁵)S(O)₂N(R^(y5a)R^(y5b)), —SR^(y5),—N(R^(y5)R^(y5a)), —NO₂, —OC(O)R⁵, —N(R^(y5))C(O)R^(y)a,—N(R^(y5))S(O)₂R^(y5a), —N(R^(y5))S(O)R^(y5a), —N(R^(y5))C(O)OR^(y5a),—N(R^(y5))C(O)N(R^(y5a)R^(y5b)), —OC(O)N(R^(y5)R^(y5a)), and C₁₋₆ alkyl;wherein C₁₋₆ alkyl is optionally substituted with one or more halogen,which are the same or different; and

each —R^(y3), —R^(y3a), —R^(y4), —R^(y4a), —R^(y5), —R^(y5a) and—R^(y5b) is independently selected from the group consisting of —H, andC₁₋₆ alkyl, wherein C₁₋₆ alkyl is optionally substituted with one ormore halogen, which are the same or different.

When -L²- is other than a single chemical bond, -L²- is even morepreferably selected from -T-, —C(O)O—, —O—, —C(O)—, —C(O)N(R′)—,—S(O)₂N(R′)—, —S(O)N(R′)—, —S(O)₂—, —S(O)—, —N(R^(y1))S(O)₂N(R^(y1a))—,—S—, —N(R^(y1))—, —OC(OR^(y1))(R^(y1a))—, —N(R^(y1))C(O)N(R^(y1a))_,—OC(O)N(R^(y1))—, C₁₋₅₀ alkyl, C₂₋₅₀ alkenyl, and C₂₋₅₀ alkynyl; wherein-T-, C₁₋₂₀ alkyl, C₂₋₂₀ alkenyl, and C₂₋₂₀ alkynyl are optionallysubstituted with one or more —R^(y2), which are the same or differentand wherein C₁₋₂₀ alkyl, C₂₋₂₀ alkenyl, and C₂₋₂₀ alkynyl are optionallyinterrupted by one or more groups selected from the group consisting of-T-, —C(O)O—, —O—, —C(O)—, —C(O)N(R^(y3))—, —S(O)₂N(R^(y3))—,—S(O)N(R^(y3))—, —S(O)₂—, —S(O)—, —N(R^(y3))S(O)₂N(R^(y3a))—, —S—,—N(R^(y3))—, —OC(OR^(y3))(R^(y3a))—, —N(R^(y3))C(O)N(R^(y3a))—, and—OC(O)N(R³)—;

—R^(y1) and —R^(y1a) are independently of each other selected from thegroup consisting of —H, -T, C₁₋₁₀ alkyl, C₂₋₁₀ alkenyl, and C₂₋₁₀alkynyl; wherein -T, C₁₋₁₀ alkyl, C₂₋₁₀ alkenyl, and C₂₋₁₀ alkynyl areoptionally substituted with one or more —R^(y2), which are the same ordifferent, and wherein C₁₋₁₀ alkyl, C₂₋₁₀ alkenyl, and C₂₋₁₀ alkynyl areoptionally interrupted by one or more groups selected from the groupconsisting of -T-, —C(O)O—, —O—, —C(O)—, —C(O)N(R^(y4))—,—S(O)₂N(R^(y4))—, —S(O)N(R^(y4))—, —S(O)₂—, —S(O)—,—N(R^(y4))S(O)₂N(R^(y4a))—, —S—, —N(R^(y4))—, —OC(OR^(y4))(R^(y4a))—,—N(R^(y4))C(O)N(R^(y4a))—, and —OC(O)N(R^(y4))—;

each T is independently selected from the group consisting of phenyl,naphthyl, indenyl, indanyl, tetralinyl, C₃₋₁₀ cycloalkyl, 3- to10-membered heterocyclyl, 8- to 11-membered heterobicyclyl, 8- to30-membered carbopolycyclyl, and 8- to 30-membered heteropolycyclyl;wherein each T is independently optionally substituted with one or more—R^(y2), which are the same or different;

—R^(y2) is selected from the group consisting of halogen, —CN, oxo (═O),—COOR^(y5), —OR^(y5), —C(O)R^(y5), —C(O)N(R^(y5)R^(y5a)),—S(O)₂N(R^(y5)R^(y5a)), —S(O)N(R^(y5)R^(y5a)), —S(O)₂R^(y5),—S(O)R^(y5), —N(R^(y5))S(O)₂N(R^(y5a)R^(y5b)), —SR^(y5),—N(R^(y5)R^(y5a)), —NO₂, —OC(O)R⁵, —N(R^(y5)) C(O)R^(y5a),—N(R^(y5))S(O)₂R^(y5a), —N(R^(y5))S(O)R^(y5a), —N(R^(y5))C(O)OR^(y5)a,—N(R^(y5))C(O)N(R^(y5a)R^(y5b)), —OC(O)N(R^(y5)R^(y5a)), and C₁₋₆ alkyl;wherein C₁₋₆ alkyl is optionally substituted with one or more halogen,which are the same or different; and

each —R^(y3), —R^(y3a), —R^(y4), —R^(y4a), —R^(y5), —R^(y5a) and—R^(y5b) is independently of each other selected from the groupconsisting of —H, and C₁₋₆ alkyl; wherein C₁₋₆ alkyl is optionallysubstituted with one or more halogen, which are the same or different.

When -L²- is other than a single chemical bond, -L²- is even morepreferably selected from the group consisting of -T-, —C(O)O—, —O—,—C(O)—, —C(O)N(R^(y1))—, —S(O)₂N(R′)—, —S(O)N(R^(y1))—, —S(O)₂—, —S(O)—,—N(R^(y1))S(O)₂N(R^(y1a))—, —S—, —N(R^(y1))—, —OC(OR^(y1))(R^(y1a))—,—N(R^(y1))C(O)N(R^(y1a))—, —OC(O)N(R^(y1))—, C₁₋₅₀ alkyl, C₂₋₅₀ alkenyl,and C₂₋₅₀ alkynyl; wherein -T-, C₁₋₅₀ alkyl, C₂₋₅₀ alkenyl, and C₂₋₅₀alkynyl are optionally substituted with one or more —R^(y2), which arethe same or different and wherein C₁₋₅₀ alkyl, C₂₋₅₀ alkenyl, and C₂₋₅₀alkynyl are optionally interrupted by one or more groups selected fromthe group consisting of -T-, —C(O)O—, —O—, —C(O)—, —C(O)N(R^(y3))—,—S(O)₂N(R^(y3))—, —S(O)N(R^(y3))—, —S(O)₂—, —S(O)—,—N(R^(y3))S(O)₂N(R^(y3a))—, —S—, —N(R^(y3))—, —OC(OR^(y3))(R^(y3a))—,—N(R^(y3))C(O)N(R^(y3a))—, and —OC(O)N(R^(y3))—;

—R^(y1) and —R^(y1a) are independently selected from the groupconsisting of —H, -T, C₁₋₁₀ alkyl, C₂₋₁₀ alkenyl, and C₂₋₁₀ alkynyl;

each T is independently selected from the group consisting of phenyl,naphthyl, indenyl, indanyl, tetralinyl, C₃₋₁₀ cycloalkyl, 3- to10-membered heterocyclyl, 8- to 11-membered heterobicyclyl, 8- to30-membered carbopolycyclyl, and 8- to 30-membered heteropolycyclyl;

each —R^(y2) is independently selected from the group consisting ofhalogen, and C₁₋₆ alkyl; and

each —R^(y3), —R^(y3a), —R^(y4), —R^(y4a), —R^(y5), —R^(y5a) and—R^(y5b) is independently of each other selected from the groupconsisting of —H, and C₁₋₆ alkyl; wherein C₁₋₆ alkyl is optionallysubstituted with one or more halogen, which are the same or different.

Even more preferably, -L²- is a C₁₋₂₀ alkyl chain, which is optionallyinterrupted by one or more groups independently selected from —O—, -T-and —C(O)N(R^(y1))—; and which C₁₋₂₀ alkyl chain is optionallysubstituted with one or more groups independently selected from —OH, -Tand —C(O)N(R^(y6)R^(y6a)); wherein —R^(y1), —R^(y6), —R^(y6a) areindependently selected from the group consisting of H and C₁₋₄ alkyl andwherein T is selected from the group consisting of phenyl, naphthyl,indenyl, indanyl, tetralinyl, C₃₋₁₀ cycloalkyl, 3- to 10-memberedheterocyclyl, 8- to 11-membered heterobicyclyl, 8- to 30-memberedcarbopolycyclyl, and 8- to 30-membered heteropolycyclyl.

Preferably, -L²- has a molecular weight in the range of from 14 g/mol to750 g/mol.

Preferably, -L²- comprises a moiety selected from

wherein

dashed lines indicate attachment to the rest of -L²-, -L¹-, —Z and/or—Z′, respectively; and

—R and —R^(a) are independently of each other selected from the groupconsisting of —H, methyl, ethyl, propyl, butyl, pentyl and hexyl.

In one preferred embodiment -L²- has a chain lengths of 1 to 20 atoms.

As used herein the term “chain length” with regard to the moiety -L²-refers to the number of atoms of -L²- present in the shortest connectionbetween -L¹- and —Z.

Preferably, -L²- is of formula (i)

-   -   wherein    -   the dashed line marked with the asterisk indicates attachment to        -L¹-;    -   the unmarked dashed line indicates attachment to —Z or —Z′;    -   —R¹ is selected from the group consisting of —H, C₁₋₆ alkyl,        C₂₋₆ alkenyl and C₂₋₆ alkynyl;    -   n is selected from the group consisting of 0, 1, 2, 3, 4, 5, 6,        7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 and 18; and    -   wherein the moiety of formula (i) is optionally further        substituted.

Preferably —R¹ of formula (i) is selected from the group consisting of—H, methyl, ethyl, propyl, and butyl. Even more preferably —R¹ offormula (i) is selected from the group consisting of —H, methyl, ethyland propyl. Even more preferably —R¹ of formula (i) is selected from thegroup consisting of —H and methyl. Most preferably —R¹ of formula (i) ismethyl.

Preferably n of formula (i) is selected from the group consisting of 0,1, 2, 3, 4, 5, 6, 7, 8, 9 and 10. Even more preferably n of formula (i)is selected from the group consisting of 0, 1, 2, 3, 4 and 5. Even morepreferably n of formula (i) is selected from the group consisting of 0,1, 2 and 3. Even more preferably n of formula (i) is selected from thegroup consisting of 0 and 1. Most preferably n of formula (i) is 0.

In one preferred embodiment -L²- is a moiety selected from the groupconsisting of

-   -   wherein    -   the dashed line marked with the asterisk indicates attachment to        -L-;    -   the unmarked dashed line indicates attachment to —Z or —Z′; and    -   wherein the moieties (ii), (iii), (iv), (v), (vi), (vii),        (viii), (ix), (x), (xi), (xii), (xiii), (xiv), (xv), (xvi)        and (xvii) are optionally further substituted.

In a preferred embodiment -L²- is selected from the group consisting of

-   -   wherein    -   the dashed line marked with the asterisk indicates attachment to        -L-; and    -   the unmarked dashed line indicates attachment to —Z or —Z′.

Even more preferred -L²- is selected from the group consisting of

-   -   wherein    -   the dashed line marked with the asterisk indicates attachment to        -L¹-; and    -   the unmarked dashed line indicates attachment to —Z or —Z′.

Even more preferably -L²- is of formula (xvi)

-   -   wherein    -   the dashed line marked with the asterisk indicates attachment to        -L¹-; and    -   the unmarked dashed line indicates attachment to —Z or —Z′.

In one preferred embodiment the moiety -L¹-L²- is selected from thegroup consisting of

-   -   wherein    -   the unmarked dashed line indicates the attachment to a nitrogen        of -D which is a CNP agonist moiety by forming an amide bond;        and    -   the dashed line marked with the asterisk indicates attachment to        —Z or —Z′.

In an even more preferred embodiment the moiety -L¹-L²- is of formula(IIc-ii)

-   -   wherein    -   the unmarked dashed line indicates the attachment to a nitrogen        of -D which is a CNP agonist moiety by forming an amide bond;        and    -   the dashed line marked with the asterisk indicates attachment to        —Z or —Z′.

In a most preferred embodiment the moiety -L -L²- is of formula(IId-ii′)

-   -   wherein    -   the unmarked dashed line indicates the attachment to a nitrogen        of -D which is a CNP agonist moiety by forming an amide bond;        and    -   the dashed line marked with the asterisk indicates attachment to        —Z or —Z′.

In another preferred embodiment the moiety -L¹-L²- is selected from thegroup consisting of

-   -   wherein    -   the unmarked dashed line indicates the attachment to a nitrogen        of -D which is a CNP agonist moiety by forming an amide bond;        and    -   the dashed line marked with the asterisk indicates attachment to        —Z or —Z′.

In an even more preferred embodiment the moiety -L-L²- is of formula(IId-iia)

-   -   wherein    -   the unmarked dashed line indicates the attachment to a nitrogen        of -D which is a CNP agonist moiety by forming an amide bond;        and    -   the dashed line marked with the asterisk indicates attachment to        —Z or —Z′.

In a most preferred embodiment the moiety -L¹-L²- is of formula(IId-iia′)

-   -   wherein    -   the unmarked dashed line indicates the attachment to a nitrogen        of -D which is a CNP agonist moiety by forming an amide bond;        and    -   the dashed line marked with the asterisk indicates attachment to        —Z or —Z′.

In another preferred embodiment the moiety -L¹-L²- is selected from thegroup consisting of

-   -   wherein    -   the unmarked dashed line indicates the attachment to a nitrogen        of -D which is a CNP agonist moiety by forming an amide bond;        and    -   the dashed line marked with the asterisk indicates attachment to        —Z or —Z′.

In an even more preferred embodiment the moiety -L¹-L²- is

-   -   wherein    -   the unmarked dashed line indicates the attachment to a nitrogen        of -D which is a CNP agonist moiety by forming an amide bond;        and    -   the dashed line marked with the asterisk indicates attachment to        —Z or —Z′.

In a most preferred embodiment the moiety -L¹-L²- is of formula(IId-iib′)

-   -   wherein    -   the unmarked dashed line indicates the attachment to a nitrogen        of -D which is a CNP agonist moiety by forming an amide bond;        and    -   the dashed line marked with the asterisk indicates attachment to        —Z or —Z′.

Preferably, —Z of formula (Ia) or (Ib) has a molecular weight rangingfrom 5 to 200 kDa. Even more preferably, —Z of formula (Ia) or (Ib) hasa molecular weight ranging from 8 to 100 kDa, even more preferablyranging from 10 to 80 kDa, even more preferably from 12 to 60, even morepreferably from 15 to 40 and most preferably —Z of formula (Ia) or (Ib)has a molecular weight of about 20 kDa. In another equally preferredembodiment —Z of formula (Ia) or (Ib) has a molecular weight of about 40kDa.

The carrier —Z of formula (Ia) or (Ib) comprises a C₈₋₂₄ alkyl or apolymer. Preferably, —Z of formula (Ia) or (Ib) comprises a polymer,preferably a polymer selected from the group consisting of2-methacryloyl-oxyethyl phosphoyl cholins, poly(acrylic acids),poly(acrylates), poly(acrylamides), poly(alkyloxy) polymers,poly(amides), poly(amidoamines), poly(amino acids), poly(anhydrides),poly(aspartamides), poly(butyric acids), poly(glycolic acids),polybutylene terephthalates, poly(caprolactones), poly(carbonates),poly(cyanoacrylates), poly(dimethylacrylamides), poly(esters),poly(ethylenes), poly(ethyleneglycols), poly(ethylene oxides),poly(ethyl phosphates), poly(ethyloxazolines), poly(glycolic acids),poly(hydroxyethyl acrylates), poly(hydroxyethyl-oxazolines),poly(hydroxymethacrylates), poly(hydroxypropylmethacrylamides),poly(hydroxypropyl methacrylates), poly(hydroxypropyloxazolines),poly(iminocarbonates), poly(lactic acids), poly(lactic-co-glycolicacids), poly(methacrylamides), poly(methacrylates),poly(methyloxazolines), poly(organophosphazenes), poly(ortho esters),poly(oxazolines), poly(propylene glycols), poly(siloxanes),poly(urethanes), poly(vinyl alcohols), poly(vinyl amines),poly(vinylmethylethers), poly(vinylpyrrolidones), silicones, celluloses,carbomethyl celluloses, hydroxypropyl methylcelluloses, chitins,chitosans, dextrans, dextrins, gelatins, hyaluronic acids andderivatives, functionalized hyaluronic acids, mannans, pectins,rhamnogalacturonans, starches, hydroxyalkyl starches, hydroxyethylstarches and other carbohydrate-based polymers, xylans, and copolymersthereof.

In another preferred embodiment, —Z of formula (Ia) or (Ib) comprises afatty acid derivate.

Preferred fatty acid derivatives are those disclosed in WO 2005/027978A2 and WO 2014/060512 A1 which are herewith incorporated by reference.

In one embodiment —Z of formula (Ia) or (Ib) comprises a protein.Preferred proteins are selected from the group consisting ofcarboxyl-terminal peptide of the chorionic gonadotropin as described inUS 2012/0035101 A1 which are herewith incorporated by reference;albumin; XTEN sequences as described in WO 2011123813 A2 which areherewith incorporated by reference; proline/alanine random coilsequences as described in WO 2011/144756 A1 which are herewithincorporated by reference; proline/alanine/serine random coil sequencesas described in WO 2008/155134 A1 and WO 2013/024049 A1 which areherewith incorporated by reference; and Fc fusion proteins.

In one embodiment —Z of formula (Ia) or (Ib) is a polysarcosine.

In another preferred embodiment —Z of formula (Ia) or (Ib) comprises apoly(N-methylglycine).

In a particularly preferred embodiment —Z of formula (Ia) or (Ib)comprises a random coil protein moiety.

In one preferred embodiment —Z of formula (Ia) or (Ib) comprises onerandom coil protein moiety.

In another preferred embodiment —Z of formula (Ia) or (Ib) comprises tworandom coil protein moieties.

In another preferred embodiment —Z of formula (Ia) or (Ib) comprisesthree random coil protein moieties.

In another preferred embodiment —Z of formula (Ia) or (Ib) comprisesfour random coil protein moieties.

In another preferred embodiment —Z of formula (Ia) or (Ib) comprisesfive random coil protein moieties.

In another preferred embodiment —Z of formula (Ia) or (Ib) comprises sixrandom coil protein moieties.

In another preferred embodiment —Z of formula (Ia) or (Ib) comprisesseven random coil protein moieties.

In another preferred embodiment —Z of formula (Ia) or (Ib) compriseseight random coil protein moieties.

Preferably such random coil protein moiety comprises at least 25 aminoacid residues and at most 2000 amino acids. Even more preferably suchrandom coil protein moiety comprises at least 30 amino acid residues andat most 1500 amino acid residues. Even more preferably such random coilprotein moiety comprises at least 50 amino acid residues and at most 500amino acid residues.

In a preferred embodiment, —Z of formula (Ia) or (Ib) comprises a randomcoil protein moiety of which at least 80%, preferably at least 85%, evenmore preferably at least 90%, even more preferably at least 95%, evenmore preferably at least 98% and most preferably at least 99% of thetotal number of amino acids forming said random coil protein moiety areselected from alanine and proline. Even more preferably, at least 10%,but less than 75%, preferably less than 65%, of the total number ofamino acid residues of such random coil protein moiety are prolineresidues. Preferably, such random coil protein moiety is as described inWO 2011/144756 A1 which is hereby incorporated by reference in itsentirety. Even more preferably —Z comprises at least one moiety selectedfrom the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ IDNO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ IDNO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:51 and SEQ IDNO:61 as disclosed in WO2011/144756 which are hereby incorporated byreference. A moiety comprising such random coil protein comprisingalanine and proline will be referred to as “PA” or “PA moiety”.

Accordingly, —Z of formula (Ia) or (Ib) comprises a PA moiety.

In an equally preferred embodiment, —Z of formula (Ia) or (Ib) comprisesa random coil protein moiety of which at least 80%, preferably at least85%, even more preferably at least 90%, even more preferably at least95%, even more preferably at least 98% and most preferably at least 99%of the total number of amino acids forming said random coil proteinmoiety are selected from alanine, serine and proline. Even morepreferably, at least 4%, but less than 40% of the total number of aminoacid residues of such random coil protein moiety are proline residues.Preferably, such random coil protein moiety is as described in WO2008/155134 A1 which is hereby incorporated by reference in itsentirety. Even more preferably —Z of formula (Ia) or (Ib) comprises atleast one moiety selected from the group consisting of SEQ ID NO:2, SEQID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ IDNO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ IDNO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ IDNO:34, SEQ ID NO:36, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ IDNO:46, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54 and SEQ ID NO:56 asdisclosed in WO 2008/155134 A1, which are hereby incorporated byreference. A moiety comprising such random coil protein moietycomprising alanine, serine and proline will be referred to as “PAS” or“PAS moiety”.

Accordingly, —Z of formula (Ia) or (Ib) comprises a PAS moiety.

In an equally preferred embodiment, —Z of formula (Ia) or (Ib) comprisesa random coil protein moiety of which at least 80%, preferably at least85%, even more preferably at least 90%, even more preferably at least95%, even more preferably at least 98% and most preferably at least 99%of the total number of amino acids forming said random coil proteinmoiety are selected from alanine, glycine and proline. A moietycomprising such random coil protein moiety comprising alanine, glycineand proline will be referred to as “PAG” or “PAG moiety”.

Accordingly, —Z of formula (Ia) or (Ib) comprises a PAG moiety.

In an equally preferred embodiment, —Z of formula (Ia) or (Ib) comprisesa random coil protein moiety of which at least 80%, preferably at least85%, even more preferably at least 90%, even more preferably at least95%, even more preferably at least 98% and most preferably at least 99%of the total number of amino acids forming said random coil proteinmoiety are selected from proline and glycine. A moiety comprising suchrandom coil protein moiety comprising proline and glycine will bereferred to as “PG” or “PG moiety”.

Preferably, such PG moiety comprises a moiety of formula (a-0)[(Gly)_(p)-Pro-(Gly)_(q)]_(r)  (a-0);

-   -   wherein    -   p is selected from the group consisting of 0, 1, 2, 3, 4 and 5;    -   q is selected from the group consisting of 0, 1, 2, 3, 4 and 5;    -   r is an integer ranging from and including 10 to 1000;    -   provided that at least one of p and q is at least 1;

Preferably, p of formula (a-0) is selected from the group consisting of1, 2 and 3.

Preferably, q of formula (a-0) is selected from 0, 1 and 2.

Even more preferably the PG moiety comprises the sequence of SEQ IDNO:97:

GGPGGPGPGGPGGPGPGGPG

Even more preferably, the PG moiety comprises the sequence of formula(a-0-a)(GGPGGPGPGGPGGPGPGGPG)_(v)  (a-0-a),

-   -   wherein    -   v is an integer ranging from and including 1 to 50.

It is understood that the sequence of formula (a-0-a) comprises vreplicates of the sequence of SEQ ID NO:97.

Accordingly, —Z of formula (Ia) or (Ib) comprises a PG moiety.

In an equally preferred embodiment, —Z of formula (Ia) or (Ib) comprisesa random coil protein moiety of which at least 80%, preferably at least85%, even more preferably at least 90%, even more preferably at least95%, even more preferably at least 98% and most preferably at least 99%of the total number of amino acids forming said random coil proteinmoiety are selected from alanine, glycine, serine, threonine, glutamateand proline. Preferably, such random coil protein moiety is as describedin WO 2010/091122 A1 which is hereby incorporated by reference. Evenmore preferably —Z of formula (Ia) or (Ib) comprises at least one moietyselected from the group consisting of SEQ ID NO:182, SEQ ID NO:183, SEQID NO:184; SEQ ID NO:185, SEQ ID NO:186, SEQ ID NO:187, SEQ ID NO:188,SEQ ID NO:189, SEQ ID NO:190, SEQ ID NO:191, SEQ ID NO:192, SEQ IDNO:193, SEQ ID NO:194, SEQ ID NO:195, SEQ ID NO:196, SEQ ID NO:197, SEQID NO:198, SEQ ID NO:199, SEQ ID NO:200, SEQ ID NO:201, SEQ ID NO:202,SEQ ID NO:203, SEQ ID NO:204, SEQ ID NO:205, SEQ ID NO:206, SEQ IDNO:207, SEQ ID NO:208, SEQ ID NO:209, SEQ ID NO:210, SEQ ID NO:211, SEQID NO:212, SEQ ID NO:213, SEQ ID NO:214, SEQ ID NO:215, SEQ ID NO:216,SEQ ID NO:217, SEQ ID NO:218, SEQ ID NO:219, SEQ ID NO:220, SEQ IDNO:221, SEQ ID NO:759, SEQ ID NO:760, SEQ ID NO:761, SEQ ID NO:762, SEQID NO:763, SEQ ID NO:764, SEQ ID NO:765, SEQ ID NO:766, SEQ ID NO:767,SEQ ID NO:768, SEQ ID NO:769, SEQ ID NO:770, SEQ ID NO:771, SEQ IDNO:772, SEQ ID NO:773, SEQ ID NO:774, SEQ ID NO:775, SEQ ID NO:776, SEQID NO:777, SEQ ID NO:778, SEQ ID NO:779, SEQ ID NO:1715, SEQ ID NO:1716,SEQ ID NO:1718, SEQ ID NO:1719, SEQ ID NO:1720, SEQ ID NO:1721 and SEQID NO:1722 as disclosed in WO2010/091122A1, which are herebyincorporated by reference. A moiety comprising such random coil proteinmoiety comprising alanine, glycine, serine, threonine, glutamate andproline will be referred to as “XTEN” or “XTEN moiety” in line with itsdesignation in WO 2010/091122 A1.

Accordingly, —Z of formula (Ia) or (Ib) comprises an XTEN moiety.

In another preferred embodiment —Z of formula (Ia) or (Ib) is ahyaluronic acid-based polymer.

In one embodiment —Z of formula (Ia) or (Ib) is a carrier as disclosedin WO 2012/02047 A1 which is herewith incorporated by reference.

In another embodiment —Z of formula (Ia) or (Ib) is a carrier asdisclosed in WO 2013/024048 A1 which is herewith incorporated byreference.

In another preferred embodiment —Z of formula (Ia) or (Ib) is aPEG-based polymer. Even more preferably —Z of formula (Ia) or (Ib) is abranched or multi-arm PEG-based polymer.

In a preferred embodiment —Z of formula (Ia) or (Ib) is branchedpolymer. In one embodiment —Z of formula (Ia) or (Ib) is a branchedpolymer having one, two, three, four, five or six branching points.Preferably, —Z of formula (Ia) or (Ib) is a branched polymer having one,two or three branching points. In one embodiment —Z of formula (Ia) or(Ib) is a branched polymer having one branching point. In anotherembodiment —Z of formula (Ia) or (Ib) is a branched polymer having twobranching points. In another embodiment —Z of formula (Ia) or (Ib) is abranched polymer having three branching points.

A branching point is preferably selected from the group consisting of—N<, —CH< and >C<.

Preferably such branched moiety —Z of formula (Ia) or (Ib) is PEG-based.

In one embodiment such branched moiety —Z of formula (Ia) or (Ib) has amolecular weight ranging from and including 5 kDa to 500 kDa, morepreferably ranging from and including 10 kDa to 250 Da, even morepreferably ranging from and including 10 kDa to 150 kDa, even morepreferably ranging from and including 12 kDa to 100 kDa and mostpreferably ranging from and including 15 kDa to 80 kDa.

Preferably, such branched moiety —Z of formula (Ia) or (Ib) has amolecular weight ranging from and including 10 kDa to 80 kDa. In oneembodiment the molecular weight is about 10 kDa. In another embodimentthe molecular weight of such branched moiety —Z of formula (Ia) or (Ib)is about 20 kDa. In another embodiment the molecular weight of suchbranched moiety —Z of formula (Ia) or (Ib) is about 30 kDa. In anotherembodiment the molecular weight of such a branched moiety —Z of formula(Ia) or (Ib) is about 40 kDa. In another embodiment the molecular weightof such a branched moiety —Z of formula (Ia) or (Ib) is about 50 kDa. Inanother embodiment the molecular weight of such a branched moiety —Z offormula (Ia) or (Ib) is about 60 kDa. In another embodiment themolecular weight of such a branched moiety —Z of formula (Ia) or (Ib) isabout 70 kDa. In another embodiment the molecular weight of such abranched moiety —Z of formula (Ia) or (Ib) is about 80 kDa. Mostpreferably, such branched moiety —Z of formula (Ia) or (Ib) has amolecular weight of about 40 kDa.

Applicants found that an N-terminal attachment of a moiety -L -L²-Z issignificantly more efficient with regard to NEP-stability thanattachment at an internal site and that the least efficient attachmentsite with regard to NEP-stability is at the ring part of a CNP moiety.However, applicants surprisingly found that this disadvantage ofattachment to the ring with regard to NEP-stability can be compensatedby using a branched moiety —Z having a molecular weight of at least 10kDa, such as at least 12 kDa, such as at least 15 kDa, such as at least18 kDa, such as at least 20 kDa, such as at least 24 kDa, such as atleast 25 kDa, such as at least 27 kDa, such as at least 30 kDa.Preferably, such branched moiety —Z has a molecular weight of no morethan 500 kDa, preferably of no more than 250 kDa, preferably of no morethan 200 Da, preferably of no more than 150 kDa and most preferably nomore than 100 kDa. Most preferably such branched moiety —Z has amolecular weight of about 40 kDa. Consequently, the use of such branchedmoiety —Z at the ring part of the CNP moiety does not only lead toincreased NEP-stability, but combines increased NEP-stability withreduced NPR-B binding associated with attachment to the ring.

It was surprisingly found that even though the ring moiety is involvedin NPR-C binding, attachment of a 5 kDa carrier to the ring moiety didnot have a significant effect on NPR-C affinity. Furthermore, it wassurprisingly found that a 4x 10 kDa carrier, i.e. a branched carrierhaving four 10 kDa arms, attached to the ring moiety is more efficientin reducing NPR-C affinity than a 2x 20 kDa carrier, i.e. a branchedcarrier having two 20 kDa arms, even though the total molecular weightwas the same. It is thus not only the total molecular weight of thecarrier attached to the ring moiety, but the particular branchingpattern of the carrier that influences NPR-C binding affinity.

This finding is also supported by the NPR-C affinity measured with a4-arm 40 kDa carrier having a different branching pattern which stillexhibited a high NPR-C affinity.

In summary, it was surprisingly found that NPR-C affinity can beefficiently reduced with a multi-branched carrier attached to the ringmoiety having a first branching point close to the CNP moiety, such asless than 300 atoms from the CNP moiety, preferably 200 atoms from theCNP moiety, even more preferably 100 atoms from the CNP moiety, evenmore preferably less than 50 atoms from the CNP moiety, even morepreferably less than 25 atoms from the CNP moiety and most preferablyless than 10 atoms from the CNP moiety.

Even more preferably, one or more further branching point(s) is/arelocated within less than 500 atoms from the CNP moiety, even morepreferably 300 atoms from the CNP moiety, even more preferably less than200 atoms from the CNP moiety, even more preferably less than 100 atomsfrom the CNP moiety, even more preferably less than 75 atoms from theCNP moiety, even more preferably less than 50 atoms from the CNP moiety,even more preferably less than 40 atoms from the CNP moiety and mostpreferably less than 35 atoms from the CNP moiety.

It was in addition also found that such branching pattern is beneficialfor in vivo stability of the CNP moiety, i.e. protection againstproteolytic degradation. It was surprisingly found that N-terminaldegradation was stronger when using a 2x 20 kDa carrier compared to 4x10 kDa carrier. Likewise, using a 4-arm 40 kDa carrier having adifferent branching pattern exhibited even stronger N-terminaldegradation.

Preferably, —Z or —Z′ comprises a moiety

In an equally preferred embodiment —Z or —Z′ comprises an amide bond.

In one embodiment —Z of formula (Ia) or (Ib) comprises a moiety offormula (a)

-   -   wherein    -   the dashed line indicates attachment to -L²- or to the remainder        of —Z;    -   BP^(a) is a branching point selected from the group consisting        of —N<, —CR< and >C<;    -   —R is selected from the group consisting of —H and C₁₋₆ alkyl;    -   a is 0 if BP^(a) is —N< or —CR< and a is 1 if BP^(a) is >C<;    -   S^(a)—, —S^(a′)—, —S^(a″)— and —S^(a′″)— are independently of        each other a chemical bond or are selected from the group        consisting of C₁₋₅₀ alkyl, C₂₋₅₀ alkenyl, and C₂₋₅₀ alkynyl;        wherein C₁₋₅₀ alkyl, C₂₋₅₀ alkenyl, and C₂₋₅₀ alkynyl are        optionally substituted with one or more —R¹, which are the same        or different and wherein C₁₋₅₀ alkyl, C₂₋₅₀ alkenyl, and C₂₋₅₀        alkynyl are optionally interrupted by one or more groups        selected from the group consisting of -T-, —C(O)O—, —O—, —C(O)—,        —C(O)N(R²)—, —S(O)₂N(R²)—, —S(O)N(R²)—, —S(O)₂—, —S(O)—,        —N(R²)S(O)₂N(R^(2a))—, —S—, —N(R²)—, —OC(OR²)(R^(2a))—,        —N(R²)C(O)N(R^(2a))—, and —OC(O)N(R²)—;    -   each -T- is independently selected from the group consisting of        phenyl, naphthyl, indenyl, indanyl, tetralinyl, C₃₋₁₀        cycloalkyl, 3- to 10-membered heterocyclyl, 8- to 11-membered        heterobicyclyl, 8- to 30-membered carbopolycyclyl, and 8- to        30-membered heteropolycyclyl; wherein each -T- is independently        optionally substituted with one or more —R¹, which are the same        or different;    -   each —R¹ is independently selected from the group consisting of        halogen, —CN, oxo (═O), —COOR³, —OR³, —C(O)R³, —C(O)N(R³R^(3a)),        —S(O)₂N(R³R^(3a)), —S(O)N(R³R^(3a)), —S(O)₂R³, —S(O)R³,        —N(R³)S(O)₂N(R^(3a)R^(3b)), —SR³, —N(R³R^(3a)), —NO₂, —OC(O)R³,        —N(R³)C(O)R^(3a), —N(R³)S(O)₂R^(3a), —N(R³)S(O)R^(3a),        —N(R³)C(O)OR^(3a), —N(R³)C(O)N(R^(3a)R^(3b)), —OC(O)N(R³R^(3a)),        and C₁₋₆ alkyl; wherein C₁₋₆ alkyl is optionally substituted        with one or more halogen, which are the same or different;    -   each —R², —R^(2a), —R³, —R^(3a) and —R^(3b) is independently        selected from the group consisting of —H, and C₁₋₆ alkyl,        wherein C₁₋₆ alkyl is optionally substituted with one or more        halogen, which are the same or different; and    -   —P^(a′), —P^(a″) and —P^(a′″) are independently a polymeric        moiety.

Optionally, the moiety of formula (a) is substituted with one or moresubstituents.

In one embodiment BP^(a) of formula (a) is —N<.

In another embodiment BP^(a) of formula (a) is —CR<. Preferably, —R is—H. Accordingly, a of formula (a) is preferably 0.

In another embodiment BP^(a) of formula (a) is >C<.

In one embodiment —S^(a)— of formula (a) is a chemical bond.

In another embodiment —S^(a)— of formula (a) is selected from the groupconsisting of C₁₋₁₀ alkyl, C₂₋₁₀ alkenyl and C₂₋₁₀ alkynyl, which C₁₋₁₀alkyl, C₂₋₁₀ alkenyl and C₂₋₁₀ alkynyl are optionally interrupted by oneor more chemical groups selected from the group consisting of —C(O)O—,—O—, —C(O)—, —C(O)N(R⁴)—, —S(O)₂N(R⁴)—, —S(O)N(R⁴)—, —S(O)₂—, —S(O)—,—N(R⁴)S(O)₂N(R^(4a))—, —S—, —N(R⁴)—, —OC(OR⁴)(R^(4a))—,—N(R⁴)C(O)N(R^(4a))—, and —OC(O)N(R⁴)—; wherein —R⁴ and —R^(4a) areindependently selected from the group consisting of —H, methyl, ethyl,propyl and butyl. Preferably —S^(a)— of formula (a) is selected from thegroup consisting of methyl, ethyl, propyl, butyl, which are optionallyinterrupted by one or more chemical groups selected from the groupconsisting of —O—, —C(O)— and —C(O)N(R⁴)—.

In one embodiment —S^(a′)— of formula (a) is a chemical bond.

In another embodiment —S^(a′)— of formula (a) is selected from the groupconsisting of C₁₋₁₀ alkyl, C₂₋₁₀ alkenyl and C₂₋₁₀ alkynyl, which C₁₋₁₀alkyl, C₂₋₁₀ alkenyl and C₂₋₁₀ alkynyl are optionally interrupted by oneor more chemical groups selected from the group consisting of —C(O)O—,—O—, —C(O)—, —C(O)N(R⁴)—, —S(O)₂N(R⁴)—, —S(O)N(R⁴)—, —S(O)₂—, —S(O)—,—N(R⁴)S(O)₂N(R^(4a))—, —S—, —N(R⁴)—, —OC(OR⁴)(R^(4a))—,—N(R⁴)C(O)N(R^(4a))—, and —OC(O)N(R⁴)—; wherein —R⁴ and —R^(4a) areindependently selected from the group consisting of —H, methyl, ethyl,propyl and butyl. Preferably —S^(a′)— of formula (a) is selected fromthe group consisting of methyl, ethyl, propyl, butyl, which areoptionally interrupted by one or more chemical groups selected from thegroup consisting of —O—, —C(O)— and —C(O)N(R⁴)—.

In one embodiment —S^(a″)— of formula (a) is a chemical bond.

In another embodiment —S^(a″)— of formula (a) is selected from the groupconsisting of C₁₋₁₀ alkyl, C₂₋₁₀ alkenyl and C₂₋₁₀ alkynyl, which C₁₋₁₀alkyl, C₂₋₁₀ alkenyl and C₂₋₁₀ alkynyl are optionally interrupted by oneor more chemical groups selected from the group consisting of —C(O)O—,—O—, —C(O)—, —C(O)N(R⁴)—, —S(O)₂N(R⁴)—, —S(O)N(R⁴)—, —S(O)₂—, —S(O)—,—N(R⁴)S(O)₂N(R^(4a))—, —S—, —N(R⁴)—, —OC(OR⁴)(R^(4a))—,—N(R⁴)C(O)N(R^(4a))—, and —OC(O)N(R⁴)—; wherein —R⁴ and —R^(4a) areindependently selected from the group consisting of —H, methyl, ethyl,propyl and butyl. Preferably —S^(a″)— of formula (a) is selected fromthe group consisting of methyl, ethyl, propyl, butyl, which areoptionally interrupted by one or more chemical groups selected from thegroup consisting of —O—, —C(O)— and —C(O)N(R⁴)—.

In one embodiment —S^(a′″)— of formula (a) is a chemical bond.

In another embodiment —S^(a′″)— of formula (a) is selected from thegroup consisting of C₁₋₁₀ alkyl, C₂₋₁₀ alkenyl and C₂₋₁₀ alkynyl, whichC₁₋₁₀ alkyl, C₂₋₁₀ alkenyl and C₂₋₁₀ alkynyl are optionally interruptedby one or more chemical groups selected from the group consisting of—C(O)O—, —O—, —C(O)—, —C(O)N(R⁴)—, —S(O)₂N(R⁴)—, —S(O)N(R⁴)—, —S(O)₂—,—S(O)—, —N(R⁴)S(O)₂N(R^(4a))—, —S—, —N(R⁴)—, —OC(OR₄)(R^(4a))—,—N(R⁴)C(O)N(R^(4a))—, and —OC(O)N(R⁴)—; wherein —R⁴ and —R^(4a) areindependently selected from the group consisting of —H, methyl, ethyl,propyl and butyl. Preferably —S^(a′″)— of formula (a) is selected fromthe group consisting of methyl, ethyl, propyl, butyl, which areoptionally interrupted by one or more chemical groups selected from thegroup consisting of —O—, —C(O)— and —C(O)N(R⁴)—.

Preferably, —P^(a′), —P^(a″) and —P^(a′″) of formula (a) independentlycomprise a polymer selected from the group consisting of2-methacryloyl-oxyethyl phosphoyl cholins, poly(acrylic acids),poly(acrylates), poly(acrylamides), poly(alkyloxy) polymers,poly(amides), poly(amidoamines), poly(amino acids), poly(anhydrides),poly(aspartamides), poly(butyric acids), poly(glycolic acids),polybutylene terephthalates, poly(caprolactones), poly(carbonates),poly(cyanoacrylates), poly(dimethylacrylamides), poly(esters),poly(ethylenes), poly(ethyleneglycols), poly(ethylene oxides),poly(ethyl phosphates), poly(ethyloxazolines), poly(glycolic acids),poly(hydroxyethyl acrylates), poly(hydroxyethyl-oxazolines),poly(hydroxymethacrylates), poly(hydroxypropylmethacrylamides),poly(hydroxypropyl methacrylates), poly(hydroxypropyloxazolines),poly(iminocarbonates), poly(lactic acids), poly(lactic-co-glycolicacids), poly(methacrylamides), poly(methacrylates),poly(methyloxazolines), poly(organophosphazenes), poly(ortho esters),poly(oxazolines), poly(propylene glycols), poly(siloxanes),poly(urethanes), poly(vinyl alcohols), poly(vinyl amines),poly(vinylmethylethers), poly(vinylpyrrolidones), silicones, celluloses,carbomethyl celluloses, hydroxypropyl methylcelluloses, chitins,chitosans, dextrans, dextrins, gelatins, hyaluronic acids andderivatives, functionalized hyaluronic acids, mannans, pectins,rhamnogalacturonans, starches, hydroxyalkyl starches, hydroxyethylstarches and other carbohydrate-based polymers, xylans, and copolymersthereof.

Preferably, —P^(a′), —P^(a″) and —P^(a′″) of formula (a) independentlyhave a molecular weight ranging from and including 5 kDa to 50 kDa, morepreferably have a molecular weight ranging from and including 5 kDa to40 kDa, even more preferably ranging from and including 7.5 kDa to 35kDa, even more preferably ranging from and 7.5 to 30 kDa, even morepreferably ranging from and including 10 to 30 kDa.

In one embodiment —P^(a′), —P^(a″) and —P^(a′″) of formula (a) have amolecular weight of about 5 kDa.

In another embodiment —P^(a′), —P^(a″) and —P^(a′″) of formula (a) havea molecular weight of about 7.5 kDa.

In another embodiment —P^(a′), —P^(a″) and —P^(a′″) of formula (a) havea molecular weight of about 10 kDa.

In another embodiment —P^(a′), —P^(a″) and —P^(a′″) of formula (a) havea molecular weight of about 12.5 kDa.

In another embodiment —P^(a′), —P^(a″) and —P^(a′″) of formula (a) havea molecular weight of about 15 kDa.

In another embodiment —P^(a′), —P^(a″) and —P^(a′″) of formula (a) havea molecular weight of about 20 kDa.

More preferably, —P^(a′), —P^(a′″) and —P^(a′″) of formula (a)independently comprise a PEG-based moiety. Even more preferably,—P^(a′), —P^(a″) and —P^(a′″) of formula (a) independently comprise aPEG-based moiety comprising at least 20% PEG, even more preferably atleast 30%, even more preferably at least 40% PEG, even more preferablyat least 50% PEG, even more preferably at least 60% PEG, even morepreferably at least 70% PEG, even more preferably at least 80% PEG andmost preferably at least 90% PEG.

In an equally preferred embodiment —P^(a′), —P^(a″) and —P^(a′″) offormula (a) independently comprise a protein moiety, more preferably arandom coil protein moiety and most preferably a random coil proteinmoiety selected from the group consisting of PA, PAS, PAG, PG and XTENmoieties.

In one embodiment —P^(a′), —P^(a′″) and —P^(a′″) of formula (a) are a PAmoiety.

In another embodiment —P^(a′), —P^(a′″) and —P^(a′″) of formula (a) area PAS moiety.

In another embodiment —P^(a′), —P^(a′″) and —P^(a′″) of formula (a) area PAG moiety.

In another embodiment —P^(a′), —P^(a′″) and —P^(a′″) of formula (a) area PG moiety.

In another embodiment —P^(a′), —P^(a′″) and —P^(a′″) of formula (a) arean XTEN moiety.

In one embodiment —Z comprises one moiety of formula (a).

In another embodiment —Z comprises two moieties of formula (a).

In another embodiment —Z comprises three moieties of formula (a).

In another embodiment —Z comprises four moieties of formula (a).

In another embodiment —Z comprises five moieties of formula (a).

In another embodiment —Z comprises six moieties of formula (a).

In a preferred embodiment —Z comprises two moieties of formula (a).

In a preferred embodiment —Z comprises a moiety of formula (b)

-   -   wherein    -   the dashed line indicates attachment to -L²- or to the remainder        of —Z;    -   b1 is selected from the group consisting of 0, 1, 2, 3, 4, 5, 6,        7 and 8;    -   b2 is selected from the group consisting of 1, 2, 3, 4, 5, 6, 7        and 8;    -   b3 is an integer ranging from and including 150 to 1000;        preferably ranging from and including 150 to 500; and most        preferably ranging from and including 200 to 460; and    -   b4 is an integer ranging from and including 150 to 1000;        preferably ranging from and including 150 to 500; and most        preferably ranging from and including 200 to 460.

Optionally, the moiety of formula (b) is substituted with one or moresubstituents.

Preferably, b3 and b4 of formula (b) are the same integer.

In one preferred embodiment b3 and b4 both an integer ranging from 200to 250 and most preferably b3 and b4 of formula (b) are about 225.

In another preferred embodiment b3 and b4 are both an integer rangingfrom 400 to 500 and most preferably b3 and b4 of formula (b) are about450.

Preferably, b1 of formula (b) is selected from the group consisting of0, 1, 2, 3 and 4. More preferably b1 of formula (b) is selected from thegroup consisting of 1, 2 and 3. Most preferably b of formula (b) is 2.

Preferably, b2 of formula (b) is selected from the group consisting of1, 2, 3, 4 and 5. More preferably b2 of formula (b) is selected from thegroup consisting of 2, 3 and 4. Most preferably b2 of formula (b) is 3.

In one particularly preferred embodiment b1 of formula (b) is 2, b2 offormula (b) is 3, and b3 and b4 are both about 450.

In another particularly preferred embodiment b1 of formula (b) is 2, b2of formula (b) is 3, and b3 and b4 are both about 225.

In one embodiment —Z comprises one moiety of formula (b).

In another embodiment —Z comprises two moieties of formula (b).

In another embodiment —Z comprises three moieties of formula (b).

In another embodiment —Z comprises four moieties of formula (b).

In another embodiment —Z comprises five moieties of formula (b).

In another embodiment —Z comprises six moieties of formula (b).

In a preferred embodiment —Z comprises two moieties of formula (b).

In an even more preferred embodiment —Z comprises a moiety of formula(c)

-   -   wherein    -   the dashed line indicates attachment to -L²- or to the remainder        of —Z;    -   c1 and c2 are independently an integer ranging from and        including 150 to 500; preferably ranging from and including 200        to 460.

Optionally, the moiety of formula (c) is substituted with one or moresubstituents.

Preferably both c1 and c2 of formula (c) are the same integer.

In one preferred embodiment c1 and c2 of formula (c) range from andinclude 200 to 250 and most preferably are about 225. In anotherpreferred embodiment c1 and c2 of formula (c) range from and include 400to 500 and most preferably are about 450.

In a preferred embodiment the moiety —Z is a branched PEG-based polymercomprising at least 10% PEG, has one branching point and two PEG-basedpolymer arms and has a molecular weight of about 40 kDa. Accordingly,each of the two PEG-based polymer arms has a molecular weight of about20 kDa. Preferably the branching point is —CH<.

In one embodiment —Z comprises one moiety of formula (c).

In another embodiment —Z comprises two moieties of formula (c).

In another embodiment —Z comprises three moieties of formula (c).

In another embodiment —Z comprises four moieties of formula (c).

In another embodiment —Z comprises five moieties of formula (c).

In another embodiment —Z comprises six moieties of formula (c).

In a preferred embodiment —Z comprises two moieties of formula (c).

In one preferred embodiment the moiety —Z is of formula (d)

-   -   wherein    -   the dashed line indicates attachment to -L²-;    -   —Z^(b)— is selected from the group consisting of C₁₋₅₀ alkyl,        C₂₋₅₀ alkenyl, and C₂₋₅₀ alkynyl; wherein C₁₋₅₀ alkyl, C₂₋₅₀        alkenyl, and C₂₋₅₀ alkynyl are optionally substituted with one        or more —R¹, which are the same or different and wherein C₁₋₅₀        alkyl, C₂₋₅₀ alkenyl, and C₂₋₅₀ alkynyl are optionally        interrupted by one or more groups selected from the group        consisting of -T-, —C(O)O—, —O—, —C(O)—, —C(O)N(R²)—,        —S(O)₂N(R²)—, —S(O)N(R²)—, —S(O)₂—, —S(O)—,        —N(R²)S(O)₂N(R^(2a))—, —S—, —N(R²)—, —OC(OR²)(R^(2a))—,        —N(R²)C(O)N(R^(2a))—, and —OC(O)N(R²)—;        -   each -T- is independently selected from the group consisting            of phenyl, naphthyl, indenyl, indanyl, tetralinyl, C₃₋₁₀            cycloalkyl, 3- to 10-membered heterocyclyl, 8- to            11-membered heterobicyclyl, 8- to 30-membered            carbopolycyclyl, and 8- to 30-membered heteropolycyclyl;            wherein each -T- is independently optionally substituted            with one or more —R¹, which are the same or different;        -   each —R¹ is independently selected from the group consisting            of halogen, —CN, oxo (═O), —COOR³, —OR³, —C(O)R³,            —C(O)N(R³R^(3a)), —S(O)₂N(R³R^(3a)), —S(O)N(R³R^(3a)),            —S(O)₂R³, —S(O)R³, —N(R³)S(O)₂N(R^(3a)R^(3b)), —SR³,            —N(R³R^(3a)), —NO₂, —OC(O)R³, —N(R³)C(O)R^(3a),            —N(R³)S(O)₂R^(3a), —N(R³)S(O)R^(3a), —N(R³)C(O)OR^(3a),            —N(R³)C(O)N(R^(3a)R^(3b)), —OC(O)N(R³R^(3a)), and C₁₋₆            alkyl;    -   wherein C₁₋₆ alkyl is optionally substituted with one or more        halogen, which are the same or different;    -   each —R², —R^(2a), —R³, —R^(3a) and —R^(3b) is independently        selected from the group consisting of —H, and C₁₋₆ alkyl,        wherein C₁₋₆ alkyl is optionally substituted with one or more        halogen, which are the same or different; and    -   —Z^(a) is

-   -   wherein    -   BP^(a), —S^(a)—, —S^(a′)—, —S^(a″)—, —S^(a′″)—, —P^(a′),        —P^(a″), —P^(a′″) and a are used as defined for formula (a).

Optionally, the moiety of formula (d) is substituted with one or moresubstituents.

Preferred embodiments of BP^(a), —S^(a)—, —S^(a′)—, —S^(a″)—, —S^(a′″)—,—P^(a′), —P^(a″), —P^(a′″) of formula (d) are as defined above forformula (a).

Preferably, —Z^(a) of formula (d) is of formula (b). Preferredembodiments of b1, b2, b3 and b4 are as described for formula (b).

Even more preferably, —Z^(a) of formula (d) is of formula (c). Preferredembodiments for c1 and c2 are as described for formula (c).

In an even more preferred embodiment the moiety —Z of formula (Ia) or(Ib) is of formula (e)

-   -   wherein    -   the dashed line indicates attachment to -L²-;    -   e is selected from the group consisting of 1, 2, 3, 4, 5, 6, 7,        8, 9, 10, 11, 12, 13, 14 and 15; and    -   —Z^(a) is

-   -   wherein b1, b2, b3 and b4 are used as defined for formula (b).    -   Optionally, the moiety of formula (e) is substituted with one or        more substituents.

Preferred embodiments for b1, b2, b3 and b4 of formula (e) are asdefined above for formula (b).

In one embodiment e of formula (e) is 1. In another embodiment e offormula (e) is 2. In another embodiment e of formula (e) is 3. Inanother embodiment e of formula (e) is 4. In another embodiment e offormula (e) is 5. In another embodiment e of formula (e) is 6. Inanother embodiment e of formula (e) is 7. In another embodiment e offormula (e) is 8. In another embodiment e of formula (e) is 9. Inanother embodiment e of formula (e) is 10. In another embodiment e offormula (e) is 11. In another embodiment e of formula (e) is 12. Inanother embodiment e of formula (e) is 13. In another embodiment e offormula (e) is 14. In another embodiment e of formula (e) is 15.

Preferably e of formula (e) is selected from the group consisting of 2,3, 4, 5, 6, 7, 8 and 9.

Even more preferably, e of formula (e) is selected from 3, 4, 5 and 6.Most preferably e of formula (e) is 5.

Preferably e of formula (e) is 5, b1 of formula (e) is 2, b2 of formula(e) is 3 and b3 and b4 of formula (e) are both about 450.

In an equally preferred embodiment the moiety —Z of formula (Ia) or (Ib)is of formula (e-i) or (e-i′):

-   -   wherein    -   the dashed line indicates attachment to -L²-, e is selected from        the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,        13, 14 and 15;    -   —Z^(a) is

-   -   wherein    -   b 1, b2, b3 and b4 are used as defined for formula (b).

Preferred embodiments for b1, b2, b3 and b4 of formula (e-i) and (e-i′)are as defined above for formula (b).

Preferred embodiments for e of formula (e-i) and (e-i′) are as describedfor formula (e).

Preferably, b1 of formula (e-i) and (e-i′) is 2, b2 of formula (e-i) and(e-i′) is 3 and b3 and b4 of formula (e-i) and (e-i′) are both about450.

In a preferred embodiment —Z of formula (Ia) or (Ib) is of formula(e-i).

In another preferred embodiment the moiety —Z is a branched PEG-basedpolymer comprising at least 10% PEG, has three branching points and fourPEG-based polymer arms and has a molecular weight of about 40 kDa.Accordingly, each of the four PEG-based polymer arms has a molecularweight of about 10 kDa. Preferably each of the three branching points is—CH<.

In a preferred embodiment the moiety —Z is of formula (f)

-   -   wherein    -   the dashed line indicates attachment to -L²-;    -   BP^(f) is a branching point selected from the group consisting        of —N<, —CR< and >C<;    -   —R is selected from the group consisting of —H and C₁₋₆ alkyl;    -   f is 0 if BP^(f) is —N< or —CR< and f is 1 if BP^(f) is >C<;    -   —S^(f)—, —S^(f′)—, —S^(f″)— and —S^(f′″)— are independently        either a chemical bond or are independently selected from the        group consisting of C₁₋₅₀ alkyl, C₂₋₅₀ alkenyl, and C₂₋₅₀        alkynyl; wherein C₁₋₅₀ alkyl, C₂₋₅₀ alkenyl, and C₂₋₅₀ alkynyl        are optionally substituted with one or more —R¹, which are the        same or different and wherein C₁₋₅₀ alkyl, C₂₋₅₀ alkenyl, and        C₂₋₅₀ alkynyl are optionally interrupted by one or more groups        selected from the group consisting of -T-, —C(O)O—, —O—, —C(O)—,        —C(O)N(R²)—, —S(O)₂N(R²)—, —S(O)N(R²)—, —S(O)₂—, —S(O)—,        —N(R²)S(O)₂N(R^(2a))—, —S—, —N(R²)—, —OC(OR²)(R^(2a))—,        —N(R²)C(O)N(R^(2a))—, and —OC(O)N(R²)—;        -   each -T- is independently selected from the group consisting            of phenyl, naphthyl, indenyl, indanyl, tetralinyl, C₃₋₁₀            cycloalkyl, 3- to 10-membered heterocyclyl, 8- to            11-membered heterobicyclyl, 8- to 30-membered            carbopolycyclyl, and 8- to 30-membered heteropolycyclyl;            wherein each -T- is independently optionally substituted            with one or more —R¹, which are the same or different;        -   each R¹ is independently selected from the group consisting            of halogen, —CN, oxo (═O), —COOR³, —OR³, —C(O)R³,            —C(O)N(R³R^(3a)), —S(O)₂N(R³R^(3a)), —S(O)N(R³R^(3a)),            —S(O)₂R³, —S(O)R³, —N(R³)S(O)₂N(R^(3a)R_(3b)), —SR³,            —N(R³R^(3a)), —NO₂, —OC(O)R³, —N(R³)C(O)R^(3a),            —N(R³)S(O)₂R^(3a), —N(R³)S(O)R^(3a), —N(R³)C(O)OR^(3a),            —N(R³)C(O)N(R^(3a)R^(3b)), —OC(O)N(R³R^(3a)), and C₁₋₆            alkyl; wherein C₁₋₆ alkyl is optionally substituted with one            or more halogen, which are the same or different;        -   each —R², —R^(2a), —R³, —R^(3a) and —R^(3b) is independently            selected from the group consisting of —H, and C₁₋₆ alkyl,            wherein C₁₋₆ alkyl is optionally substituted with one or            more halogen, which are the same or different; and    -   —Z^(a′), —Z^(a″) and —Z^(a′″) are independently

-   -   wherein    -   BP^(a), —S^(a)—, —S^(a′)—, —S^(a″)—, —S^(a′″)—, —P^(a′),        —P^(a″), —P^(a′″) and a are used as defined for formula (a).

Optionally, the moiety of formula (f) is substituted with one or moresubstituents.

Preferred embodiments of BP^(a), —S^(a)—, —S^(a′)—, —S^(a″)—, —S^(a′″)—,—P^(a′), —P^(a″) and —P^(a′″) of formula (f) are as defined above forformula (a).

Preferably BP^(f) of formula (f) is —CR< and r is 0. Preferably —R is—H.

Preferably —S^(f)— of formula (f) is a chemical bond.

Preferably, —Z^(a′), —Z^(a″) and —Z^(a′″) of formula (f) have the samestructure. Preferably, —Z^(a′), —Z^(a″) and —Z^(a′″) of formula (f) areof formula (b).

Preferred embodiments of b1, b2, b3 and b4 are as described for formula(b).

Preferably —S^(f) of formula (f) is a chemical bond, BP^(a) of formula(f) is —CR< with —R being —H. Even more preferably —S^(f)— of formula(f) is a chemical bond, BP^(a) of formula (f) is —CR< with —R being —Hand —Z^(a′), —Z^(a″) and —Z^(a′″) of formula (f) are of formula (b).

Even more preferably —Z is of formula (g)

-   -   wherein    -   the dashed line indicates attachment to -L²-; —S^(g)—, —S^(g′)—        and —S^(g′″)— are independently selected from the group        consisting of C₁₋₅₀ alkyl, C₂₋₅₀ alkenyl, and C₂₋₅₀ alkynyl;        wherein C₁₋₅₀ alkyl, C₂₋₅₀ alkenyl, and C₂₋₅₀ alkynyl are        optionally substituted with one or more —R¹, which are the same        or different and wherein C₁₋₅₀ alkyl, C₂₋₅₀ alkenyl, and C₂₋₅₀        alkynyl are optionally interrupted by one or more groups        selected from the group consisting of -T-, —C(O)O—, —O—, —C(O)—,        —C(O)N(R²)—, —S(O)₂N(R²)—, —S(O)N(R²)—, —S(O)₂—, —S(O)—,        —N(R²)S(O)₂N(R^(2a))—, —S—, —N(R²)—, —OC(OR²)(R^(2a))—,        —N(R²)C(O)N(R^(2a))—, and —OC(O)N(R²)—;        -   each -T- is independently selected from the group consisting            of phenyl, naphthyl, indenyl, indanyl, tetralinyl, C₃₋₁₀            cycloalkyl, 3- to 10-membered heterocyclyl, 8- to            11-membered heterobicyclyl, 8- to 30-membered            carbopolycyclyl, and 8- to 30-membered heteropolycyclyl;            wherein each -T- is independently optionally substituted            with one or more —R¹, which are the same or different;        -   each R¹ is independently selected from the group consisting            of halogen, —CN, oxo (═O), —COOR³, —OR³, —C(O)R³,            —C(O)N(R³R^(3a)), —S(O)₂N(R³R^(3a)), —S(O)N(R³R^(3a)),            —S(O)₂R³, —S(O)R³, —N(R³)S(O)₂N(R^(3a)R^(3b)), —SR³,            —N(R³R^(3a)), —NO₂, —OC(O)R³, —N(R³)C(O)R^(3a),            —N(R³)S(O)₂R^(3a), —N(R³)S(O)R^(3a), —N(R³)C(O)OR^(3a),            —N(R³)C(O)N(R^(3a)R^(3b)), —OC(O)N(R³R^(3a)), and C₁₋₆            alkyl; wherein C₁₋₆ alkyl is optionally substituted with one            or more halogen, which are the same or different;        -   each —R², —R^(2a), —R³, —R^(3a) and —R^(3b) is independently            selected from the group consisting of —H, and C₁₋₆ alkyl,            wherein C₁₋₆ alkyl is optionally substituted with one or            more halogen, which are the same or different; and    -   —Z^(a) and —Z^(a′) are independently

-   -   -   wherein        -   BP^(a), —S^(a)—, —S^(a′)—, —S^(a″)—, —S^(a′″)—, —P^(a′),            —P^(a″), —P^(a′″) and a are used as defined for formula (a).

Optionally, the moiety of formula (g) is substituted with one or moresubstituents.

Preferred embodiments of BP^(a), —S^(a)—, —S^(a′)—, —S^(a″)—, —S^(a′″)—,—P^(a′), —P^(a″) and —P^(a′″) of formula (g) are as defined above forformula (a).

Preferably, —S^(g)— of formula (g) is selected from the group consistingof C₁₋₆ alkyl, C₂₋₆ alkenyl and C₂₋₆ alkynyl, which are optionallysubstituted with one or more —R¹, which is the same or different,

-   -   wherein    -   R¹ is selected from the group consisting of halogen, oxo (═O),        —COOR³, —OR³, —C(O)R³, —C(O)N(R³R^(3a)), —S(O)₂N(R³R^(3a)),        —S(O)N(R³R^(3a)), —S(O)₂R³, —S(O)R³, —N(R³)S(O)₂N(R^(3a)R^(3b)),        —SR³, —N(R³R^(3a)), —NO₂, —OC(O)R³, —N(R³)C(O)R^(3a),        —N(R³)S(O)₂R^(3a), —N(R³)S(O)R^(3a), —N(R³)C(O)OR^(3a),        —N(R³)C(O)N(R^(3a)R^(3b)), —OC(O)N(R³R^(3a)), and C₁₋₆ alkyl;        wherein C₁₋₆ alkyl is optionally substituted with one or more        halogen, which are the same or different; and —R³, —R^(3a) and        —R^(3b) are independently selected from —H, methyl, ethyl,        propyl and butyl.

Even more preferably —S^(g)— of formula (g) is selected from C₁₋₆ alkyl.

Preferably, —S^(g′)— of formula (g) is selected from the groupconsisting of C₁₋₆ alkyl, C₂₋₆ alkenyl and C₂₋₆ alkynyl, which areoptionally substituted with one or more —R¹, which is the same ordifferent,

-   -   wherein    -   —R¹ is selected from the group consisting of halogen, oxo (═O),        —COOR³, —OR³, —C(O)R³, —C(O)N(R³R^(3a)), —S(O)₂N(R³R^(3a)),        —S(O)N(R³R^(3a)), —S(O)₂R³, —S(O)R³, —N(R³)S(O)₂N(R^(3a)R^(3b)),        —SR³, —N(R³R^(3a)), —NO₂, —OC(O)R³, —N(R³)C(O)R^(3a),        —N(R³)S(O)₂R^(3a), —N(R³)S(O)R^(3a), —N(R³)C(O)OR^(3a),        —N(R³)C(O)N(R^(3a)R^(3b)), —OC(O)N(R³R^(3a)), and C₁₋₆ alkyl;        wherein C₁₋₆ alkyl is optionally substituted with one or more        halogen, which are the same or different; and —R³, —R^(3a) and        —R^(3b) are independently selected from —H, methyl, ethyl,        propyl and butyl.

Even more preferably —S^(g′)— of formula (g) is selected from C₁₋₆alkyl.

Preferably, —S^(g″)— of formula (g) is selected from the groupconsisting of C₁₋₆ alkyl, C₂₋₆ alkenyl and C₂₋₆ alkynyl, which areoptionally substituted with one or more —R¹, which is the same ordifferent,

-   -   wherein    -   —R¹ is selected from the group consisting of halogen, oxo (═O),        —COOR³, —OR³, —C(O)R³, —C(O)N(R³R^(3a)), —S(O)₂N(R³R^(3a)),        —S(O)N(R³R^(3a)), —S(O)₂R³, —S(O)R³, —N(R³)S(O)₂N(R^(3a)R^(3b)),        —SR³, —N(R³R^(3a)), —NO₂, —OC(O)R³, —N(R³)C(O)R^(3a),        —N(R³)S(O)₂R^(3a), —N(R³)S(O)R^(3a), —N(R³)C(O)OR^(3a),        —N(R³)C(O)N(R^(3a)R^(3b)), —OC(O)N(R³R^(3a)), and C₁₋₆ alkyl;        wherein C₁₋₆ alkyl is optionally substituted with one or more        halogen, which are the same or different; and —R³, —R^(3a) and        —R^(3b) are independently selected from —H, methyl, ethyl,        propyl and butyl.

Even more preferably —S^(g″)— of formula (g) is selected from C₁₋₆alkyl.

Preferably, —Z^(a) and —Z^(a) of formula (g) have the same structure.Preferably, —Z^(a) and —Z^(a′) of formula (g) are of formula (b).

In an alternative even more preferred embodiment —Z of formula (Ia) or(Ib) is of formula (g-i)

-   -   wherein    -   the dashed line indicates attachment to -L²-;    -   S^(g)—, —S^(g′)— and —S^(g″)— are independently selected from        the group consisting of C₁₋₅₀ alkyl, C₂₋₅₀ alkenyl, and C₂₋₅₀        alkynyl; wherein C₁₋₅₀ alkyl, C₂₋₅₀ alkenyl, and C₂₋₅₀ alkynyl        are optionally substituted with one or more —R¹, which are the        same or different and wherein C₁₋₅₀ alkyl, C₂₋₅₀ alkenyl, and        C₂₋₅₀ alkynyl are optionally interrupted by one or more groups        selected from the group consisting of -T-, —C(O)O—, —O—, —C(O)—,        —C(O)N(R²)—, —S(O)₂N(R²)—, —S(O)N(R²)—, —S(O)₂—, —S(O)—,        —N(R²)S(O)₂N(R^(2a))—, —S—, —N(R²)—, —OC(OR²)(R^(2a))—,        —N(R²)C(O)N(R^(2a))—, and —OC(O)N(R²)—;        -   each -T- is independently selected from the group consisting            of phenyl, naphthyl, indenyl, indanyl, tetralinyl, C₃₋₁₀            cycloalkyl, 3- to 10-membered heterocyclyl, 8- to            11-membered heterobicyclyl, 8- to 30-membered            carbopolycyclyl, and 8- to 30-membered heteropolycyclyl;            wherein each -T- is independently optionally substituted            with one or more —R¹, which are the same or different;        -   each R¹ is independently selected from the group consisting            of halogen, —CN, oxo (═O), —COOR³, —OR³, —C(O)R³,            —C(O)N(R³R^(3a)), —S(O)₂N(R³R^(3a)), —S(O)N(R³R^(3a)),            —S(O)₂R³, —S(O)R³, —N(R³)S(O)₂N(R^(3a)R^(3b)), —SR³,            —N(R³R^(3a)), —NO₂, —OC(O)R³, —N(R³)C(O)R^(3a),            —N(R³)S(O)₂R^(3a), —N(R³)S(O)R^(3a), —N(R³)C(O)OR³a,            —N(R³)C(O)N(R^(3a)R^(3b)), —OC(O)N(R³R^(3a)), and C₁₋₆            alkyl; wherein C₁₋₆ alkyl is optionally substituted with one            or more halogen, which are the same or different;        -   each —R², —R^(2a), —R³, —R^(3a) and —R^(3b) is independently            selected from the group consisting of —H, and C₁₋₆ alkyl,            wherein C₁₋₆ alkyl is optionally substituted with one or            more halogen, which are the same or different;    -   —Y^(a1) and —Y^(a1′)— are

-   -   and    -   —Z^(a) and —Z^(a′) are independently

-   -   -   wherein        -   BP^(a), —S^(a)—, —S^(a′)—, —S^(a″)—, —S^(a′″)—, —P^(a′),            —P^(a″), —P^(a′″) and a are used as defined for formula (a).

Optionally, the moiety of formula (g-i) is substituted with one or moresubstituents.

Preferably, —Y^(a1)— and —Y^(a1′)— of formula (g-i) are both

wherein the dashed line marked with the asterisk is attached to —Z^(a)or —Z^(a′), respectively.

Preferred embodiments of BP^(a), —S^(a)—, —S^(a′)—, —S^(a″)—, —S^(a′″)—,—P^(a′), —P^(a″) and —P^(a′″) of formula (g-i) are as defined above forformula (a).

Preferred embodiments of —S^(g)—, —S^(g′)— and —S^(g″)— of formula (g-i)are as defined for formula (g).

Preferably, —Z^(a) and —Z^(a′) of formula (g-i) have the same structure.Preferably, —Z^(a) and —Z^(a′) of formula (g-i) are of formula (b).Preferred embodiments for b1, b2, b3 and b4 are as described for formula(b).

Even more preferably —Z is of formula (h)

-   -   wherein    -   the dashed line indicates attachment to -L²-; and    -   each —Z^(c) is a moiety

-   -   -   wherein

    -   each c1 is an integer independently ranging from about 200 to        250.

Optionally, the moiety of formula (h) is substituted with one or moresubstituents.

Preferably both c1 of formula (h) are the same.

Preferably both c1 of formula (h) are about 225.

Even more preferably —Z of formula (Ia) or (Ib) is of formula (h-a)

-   -   wherein    -   the dashed line indicates attachment to -L²-;    -   each k is independently of each other selected from the group        consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 and 12;    -   —Y^(a1)— and —Y^(a1′)— are

-   -   and    -   each —Z^(c) is a moiety

-   -   -   wherein        -   each c1 is an integer independently ranging from about 200            to 250.

Optionally, the moiety of formula (h-a) is substituted with one or moresubstituents.

Preferably, each k of formula (h-a) is independently selected from thegroup consisting of 2, 3, 4, 5, 6 and 7. Preferably, both k of formula(h-a) are identical.

Preferably both c1 of formula (h-a) are the same.

Preferably both c1 of formula (h-a) are about 225.

Preferably, —Y^(a1)— and —Y^(a1′)— of formula (h-a) are both

wherein the dashed line marked with the asterisk is attached to —Z^(c).

In an even more preferred embodiment the moiety —Z is of formula (h-i)

-   -   wherein    -   the dashed line indicates attachment to -L²-; and    -   each —Z^(c) is a moiety

-   -   each c1 is an integer independently ranging from 200 to 250.

Optionally, the moiety of formula (h-i) is substituted with one or moresubstituents.

Preferably both c1 of formula (h-i) are the same.

Preferably both c1 of formula (h-i) are about 225.

In an alternative even more preferred embodiment the moiety —Z offormula (Ia) or (Ib) is of formula (h-ia)

-   -   wherein    -   the dashed line indicates attachment to -L²-;    -   each k is independently of each other selected from the group        consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 and 12;    -   —Y^(a1)— and —Y^(a1′)— are

-   -   and    -   each —Z^(c) is a moiety

each c1 is an integer independently ranging from 200 to 250.

Preferably, each k of formula (h-ia) is independently selected from thegroup consisting of 2, 3, 4, 5, 6 and 7. Preferably, both k of formula(h-ia) are identical.

Preferably both c1 of formula (h-ia) are the same.

Preferably both c1 of formula (h-ia) are about 225.

Preferably, —Y^(a1)— and —Y^(a1′)— of formula (h-ia) are both

wherein the dashed line marked with the asterisk is attached to —Z^(c).

In an equally preferred the embodiment —Z of formula (Ia) or (Ib)comprises a moiety selected from the group consisting of

wherein

the dashed line indicates attachment to -L²-;

s1, s2, s3, s4, s5, s6, s7, s8, s9, s10, s11, s12, s13, s14 and s15 areindependently of each other selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10;

—X^(d1), —X^(d2), —X^(d3) and —X^(d4) are independently of each otherselected from the group consisting of —OH, —SH and —NR^(g1)R^(g2);preferably —OH;

—X^(e1), —X^(e2), —X^(e3) and —X^(e4) are independently of each otherselected from the group consisting of —H, C₁₋₆ alkyl, C₂₋₆ alkenyl andC₂₋₆ alkynyl;

—R^(g1) and —R^(g2) are independently of each other selected from thegroup consisting of —H, C₁₋₆ alkyl, C₂₋₆ alkenyl and C₂₋₆ alkynyl;

—X^(f1), —X^(f2), —X^(f3), —X^(f4), —X^(f5), —X^(f6), —X^(f7), —X^(f8),—X^(f9), —X^(f10), —X^(f11), —X^(f12), X^(f13) and —X^(f14) areindependently of each other selected from the group consisting of —H,C₁₋₆ alkyl, C₂₋₆ alkenyl and C₂₋₆ alkynyl; preferably —H;

-Y^(d1)_, —Y^(d2)—, —Y^(d3)— and —Y^(d4)— are independently of eachother selected from the group consisting of

and

—Z^(d1), —Z^(d2), —Z^(d3) and —Z^(d4) are independently of each other aprotein, more preferably a random coil protein and most preferably arandom coil protein selected from the group consisting of PA, PAS, PAG,PG and XTEN.

In one preferred embodiment, —Y^(d1)— and —Y^(d2)— of formula (j-iv),(j-v) and (j-vi) and —Y^(d1)—, —Y^(d2)—, —Y^(d3)— and —Y^(d4)— offormula (j-vii) are

In another preferred embodiment, —Y^(d1)— and —Y^(d2)— of formula(j-iv), (j-v) and (j-vi) and —Y^(d1)—, —Y^(d2)—, —Y^(d3)— and —Y^(d4)—of formula (j-vii) are

wherein the dashed line marked with the asterisk is oriented towards—Z^(d1), —Z^(d2), —Z^(d3) and —Z^(d4), respectively, and the unmarkeddashed line is oriented towards -L²-.

Preferably, —X^(f1), —X^(f2), —X^(f3), —X^(f4), —X^(f5), —X^(f6),—X^(f7) and —X^(f8) of formula (j-i) are —H; —X^(d1) and —X^(d2) offormula (j-i) are —OH; —X^(e1) and —X^(e2) of formula (j-i) are selectedfrom the group consisting of —H and methyl; and s1, s2, s3 and s4 offormula (j-i) are selected from the group consisting of 2, 3, 4, 5 and6. Even more preferably —X^(f1), —X^(f2), —X^(f3), —X^(f4), —X^(f5),—X^(f6), —X^(f7) and —X^(f8) of formula (j-i) are —H; —X^(d1) and—X^(d2) of formula (j-i) are —OH; —X^(e1) and —X^(e2) of formula (j-i)are —H; and s1, s2, s3 and s4 of formula (j-i) are 4.

Preferably, —X^(f1), —X^(f2), —X^(f3) and —X^(f4) of formula (j-ii) are—H; —X^(d1), —X^(d2), —X^(d3) and —X^(d2) of formula (j-ii) are —OH;—X^(e1), —X^(e2), —X^(e3) and —X^(e4) of formula (j-ii) are selectedfrom the group consisting of —H and methyl; s1, s2, s3, s4 and s5 offormula (j-ii) are selected from the group consisting of 1, 2, 3, 4, 5and 6. Even more preferably —X^(f1), —X^(f2), —X^(f3) and —X^(f4) offormula (j-ii) are —H; —X^(d1), —X^(d2), —X^(d3) and —X^(d2) of formula(j-ii) are —OH; —X^(e1), —X^(e2), —X^(e3) and —Xe⁴ of formula (j-ii) are—H; s1 is 4 of formula (j-ii) and s2, s3, s4 and s5 of formula (j-ii)are 1.

Preferably, —X^(f1), —X^(f2), —X^(f3), —X^(f4), —X^(f5), —X^(f6),—X^(f7), —X^(f8), —X^(f9) and —X^(f10) of formula (j-iii) are —H;—X^(d1), —X^(d2), —X^(d3) and —X^(d4) of formula (j-iii) are —OH;—X^(e1), —X^(e2), —X^(e3) and —X^(e4) of formula (j-iii) are selectedfrom the group consisting of —H and methyl; and s1, s2 and s3 of formula(j-iii) are selected from the group consisting of 2, 3, 4, 5 and 6. Evenmore preferably —X^(f1), —X^(f2), —X^(f3), —X^(f4), —X^(f5), —X^(f6),—X^(f7), —X^(f8), —X^(f9) and —X^(f10) of formula (j-iii) are —H;—X^(d1), —X^(d2), —X^(d3) and —X^(d4) of formula (j-iii) are —OH;—X^(e1), —X^(e2), —X^(e3) and —X^(e4) of formula (j-iii) are —H; and s1,s2 and s3 of formula (j-iii) are 4.

Preferably, —X^(f1), —X^(f2), —X^(f3), —X^(f4), —X^(f5) and —X^(f6) offormula (j-iv) are —H; s1, s2, s3, s4, s5, s6 and s7 of formula (j-iv)are selected from the group consisting of 1, 2, 3, 4, 5, 6 and 7;—Y^(d1)— and —Y^(d2)— are selected from the group consisting of

In an even more preferred embodiment —X^(f1), —X_(f2), —X^(f3), —X^(f4),—X^(f5) and —X^(f6) of formula (j-iv) are —H; s1 of formula (j-iv) is 3,s2 of formula (j-iv) is 5, s3 of formula (j-iv) is 2, s4 of formula(j-iv) is 4, s5 of formula (j-iv) is 5, s6 of formula (j-iv) is 2 and s7of formula (j-iv) is 4; and —Y^(d1)— and —Y^(d2)— of formula (j-iv) are

In an equally preferred embodiment —X^(f1), —X^(f2), —X^(f3), —X^(f4),—X^(f5) and —X^(f6) of formula (j-iv) are —H; s1 of formula (j-iv) is 3,s2 of formula (j-iv) is 5, s3 of formula (j-iv) is 2, s4 of formula(j-iv) is 4, s5 of formula (j-iv) is 5, s6 of formula (j-iv) is 2 and s7of formula (j-iv) is 4; and —Y^(d1)— and —Y^(d2)— of formula (j-iv) are

wherein the dashed line marked with the asterisk is oriented towards—Z^(d1), —Z^(d2), —Z^(d3) and —Z^(d4), respectively, and the unmarkeddashed line is oriented towards -L²-.

Preferably, —X^(f1), —X^(f2), —X^(f3) and —X^(f4) of formula (j-v) are—H; s1, s2, s3, s4 and s5 of formula (j-v) are selected from the groupconsisting of 1, 2, 3, 4, 5, 6 and 7; —Y^(d1)— and —Y^(d2)— of formula(j-v) are selected from the group consisting of

In an even more preferred embodiment —X^(f1), —X^(f2), —X^(f3) and—X^(f4) of formula (j-v) are —H; s1 of formula (j-v) is 3, s2 of formula(j-v) is 2, s3 of formula (j-v) is 1, s4 of formula (j-v) is 2 and s5 offormula (j-v) is 1; and —Y^(d1)— and -Y^(d2)— of formula (j-v) are

In an equally preferred embodiment —X^(f1), —X^(f2), —X^(f3) and —X^(f4)of formula (j-v) are —H; s1 of formula (j-v) is 3, s2 of formula (j-v)is 2, s3 of formula (j-v) is 1, s4 of formula (j-v) is 2 and s5 offormula (j-v) is 1; and —Y^(d1)— and —Y^(d2)— of formula (j-v) are

wherein the dashed line marked with the asterisk is oriented towards—Z^(d1), —Z^(d2), —Z^(d3) and —Z^(d4), respectively, and the unmarkeddashed line is oriented towards -L²-.

Preferably, —X^(f1), —X^(f2), —X^(f3), —X^(f4), —X^(f5), —X^(f6),—X^(f7), —X^(f8), —X^(f9) and —X^(f10) of formula (j-vi) are —H; s1, s2,s3, s4, s5, s6, s7, s8 and s9 of formula (j-vi) are selected from thegroup consisting of 1, 2, 3, 4, 5, 6 and 7; —Y^(d1)— and —Y^(d2)— offormula (j-vi) are selected from the group consisting of

In an even more preferred embodiment —X^(f1), —X^(f2), —X^(f3), X^(f4),—X^(f5), X^(f6), —X^(f7), —X^(f8), —X^(f9) and —X^(f10) of formula(j-vi) are —H; s1 of formula (j-vi) is 4, s2 of formula (j-vi) is 5, s3of formula (j-vi) is 2, s4 of formula (j-vi) is 4, s5 of formula (j-vi)is 4, s6 of formula (j-vi) is 5, s7 of formula (j-vi) is 2, s8 offormula (j-vi) is 4 and s9 of formula (j-vi) is 4; and —Y^(d1)— and—Y^(d2)— of formula (j-v) are

In an equally preferred embodiment —X^(f1), —X^(f2), —X^(f3), —X^(f4),—X^(f5), —X^(f6), —X^(f7), —X^(f8), —X^(f9) and —X^(f10) of formula(j-vi) are —H; s1 of formula (j-vi) is 4, s2 of formula (j-vi) is 5, s3of formula (j-vi) is 2, s4 of formula (j-vi) is 4, s5 of formula (j-vi)is 4, s6 of formula (j-vi) is 5, s7 of formula (j-vi) is 2, s8 offormula (j-vi) is 4 and s9 of formula (j-vi) is 4; and —Y^(d1)— and—Y^(d2)— of formula (j-v) are

wherein the dashed line marked with the asterisk is oriented towards—Z^(d1), —Z^(d2), —Z^(d3) and —Z^(d4), respectively, and the unmarkeddashed line is oriented towards -L²-.

Preferably, —X^(f1), —X^(f2), —X^(f3), —X^(f4), —X^(f5), —X^(f6),—X^(f7), —X^(f8), —X^(f9), —X^(f10), —X^(f11), —X^(f12), —X^(f13) and—X^(f14) of formula (j-vii) are —H; s1, s2, s3, s4, s5, s6, s7, s8, s9,s10, s11, s12, s13, s14 and s15 of formula (j-vii) are selected from thegroup consisting of 1, 2, 3, 4, 5, 6 and 7; —Y^(d1)—, —Y^(d2)—, —Y^(d3)—and —Y^(d4)— of formula (j-vii) are selected from the group consistingof

In an even more preferred embodiment —X^(f1), —X^(f2), —X^(f3), —X^(f4),—X^(f5), —X^(f6), —X^(f7), —X^(f8), —X^(f9), —X^(f10), —X^(f11),—X^(f12), —X^(f13) and —X^(f14) of formula (j-vii) are —H; are —H; s1 offormula (j-vii) is 4, s2 of formula (j-vii) is 4, s3 of formula (j-vii)is 5, s4 of formula (j-vii) is 2, s5 of formula (j-vii) is 4, s6 offormula (j-vii) is 5, s7 of formula (j-vii) is 2, s8 of formula (j-vii)is 4, s9 of formula (j-vii) is 4, s10 of formula (j-vii) is 5, s11 offormula (j-vii) is 2, s12 of formula (j-vii) is 4, s13 of formula(j-vii) is 5, s14 of formula (j-vii) is 2 and s15 of formula (j-vii) is4; and —Y^(d1)—, —Y^(d2)—, —Y^(d3)— and —Y^(d4)— of formula (j-vii) are

In an equally preferred embodiment —X^(f1), —X^(f2), —X^(f3), —X^(f4),—X^(f5), —X^(f6), —X^(f7), —X^(f8), —X^(f9), —X^(f10), —X^(f11),—X^(f12), —X^(f13) and —X^(f14) of formula (j-vii) are —H; are —H; s1 offormula (j-vii) is 4, s2 of formula (j-vii) is 4, s3 of formula (j-vii)is 5, s4 of formula (j-vii) is 2, s5 of formula (j-vii) is 4, s6 offormula (j-vii) is 5, s7 of formula (j-vii) is 2, s8 of formula (j-vii)is 4, s9 of formula (j-vii) is 4, s10 of formula (j-vii) is 5, s11 offormula (j-vii) is 2, s12 of formula (j-vii) is 4, s13 of formula(j-vii) is 5, s14 of formula (j-vii) is 2 and s15 of formula (j-vii) is4; and —Y^(d1)—, —Y^(d2)—, —Y^(d3)— and —Y^(d4)— of formula (j-vii) are

wherein the dashed line marked with the asterisk is oriented towards—Z^(d1), —Z^(d2), —Z^(d3) and —Z^(d4), respectively, and the unmarkeddashed line is oriented towards -L²-.

Preferably —Z^(d1), —Z^(d2), —Z^(d3) and —Z^(d4) of formula (j-i),(j-ii), (j-iii), (j-iv), (j-v), (j-vi) and (j-vii) have the samestructure.

In one embodiment —Z^(d1), —Z^(d2), —Z^(d3) and —Z^(d4) of formula(j-i), (j-ii), (j-iii), (j-iv), (j-v), (j-vi) and (j-vii) are a PAmoiety.

In another embodiment —Z^(d1), —Z^(d2), —Z^(d3) and —Z^(d4) of formula(j-i), (j-ii), (j-iii), (j-iv), (j-v), (j-vi) and (j-vii) are a PASmoiety.

In another embodiment —Z^(d1), —Z^(d2), —Z^(d3) and —Z^(d4) of formula(j-i), (j-ii), (j-iii), (j-iv), (j-v), (j-vi) and (j-vii) are a PAGmoiety.

In another embodiment —Z^(d1), —Z^(d2), —Z^(d3) and —Z^(d4) of formula(j-i), (j-ii), (j-iii), (j-iv), (j-v), (j-vi) and (j-vii) are a PGmoiety.

In another embodiment —Z^(d1), —Z^(d2), —Z^(s3) and —Z^(d4) of formula(j-i), (j-ii), (j-iii), (j-iv), (j-v), (j-vi) and (j-vii) are a XTENmoiety.

In a preferred embodiment the CNP agonist prodrug of the presentinvention is of formula (IIe)

-   -   wherein    -   the unmarked dashed line indicates the attachment to a nitrogen        of -D which is a CNP agonist moiety by forming an amide bond;        and    -   the dashed line marked with the asterisk indicates attachment to        a moiety

-   -   wherein    -   each c1 is an integer independently ranging from 400 to 500.

Preferably, c1 of formula (IIe) is about 450.

In an equally preferred embodiment the CNP agonist prodrug of thepresent invention is of formula (IIe-i)

-   -   wherein    -   the unmarked dashed line indicates the attachment to a nitrogen        of -D which is a CNP agonist moiety by forming an amide bond;        and    -   the dashed line marked with the asterisk indicates attachment to        a moiety

-   -   -   wherein        -   each c1 is an integer independently ranging from 400 to 500.

Preferably, c1 of formula (IIe-i) is about 450.

In another equally preferred embodiment the CNP agonist prodrug of thepresent invention is of formula (IIe-ii)

-   -   wherein    -   the unmarked dashed line indicates the attachment to a nitrogen        of -D which is a CNP agonist moiety by forming an amide bond;        and    -   the dashed line marked with the asterisk indicates attachment to        a moiety

-   -   -   wherein        -   each c1 is an integer independently ranging from 400 to 500.

Preferably, c1 of formula (IIe-ii) is about 450.

Preferably -D of formula (IIe), (IIe-i) and (IIe-ii) is a CNP moiety,i.e. the prodrug of formula (IIe), (II-i) and (IIe-ii) is a CNP prodrug.Even more preferably -D of formula (IIe), (IIe-i) and (IIe-ii) is a CNPmoiety having the sequence of SEQ ID NO:24, SEQ ID NO:25 or SEQ IDNO:30. Most preferably -D of formula ((IIe), (IIe-i) and (IIe-ii) is aCNP moiety CNP having the sequence of SEQ ID NO:24. It is also preferredthat D of formula ((IIe), (IIe-i) and (IIe-ii) is a CNP moiety CNPhaving the sequence of SEQ ID NO:20. D of formula ((IIe), (IIe-i) and(IIe-ii) is a CNP moiety CNP having the sequence of SEQ ID NO:21. D offormula ((IIe), (IIe-i) and (IIe-ii) is a CNP moiety CNP having thesequence of SEQ ID NO:22. D of formula ((IIe), (IIe-i) and (IIe-ii) is aCNP moiety CNP having the sequence of SEQ ID NO:23. D of formula ((IIe),(IIe-i) and (IIe-ii) is a CNP moiety CNP having the sequence of SEQ IDNO:30.

In one embodiment -D of formula (IIe), (IIe-i) and (IIe-ii) is a CNPmoiety which is attached to -L¹- through the nitrogen of the N-terminalamine functional group of CNP.

In a preferred embodiment -D of formula (IIe), (IIe-i) and (IIe-ii) is aCNP moiety which is attached to -L¹- through a nitrogen provided by theamine functional group of a lysine side chain of the CNP moiety.

In one embodiment said lysine side chain is not part of the ring formedby the disulfide bridge between the cysteine residues at positions 22and 38, if the CNP moiety is of SEQ ID NO:24.

Accordingly, in one embodiment the CNP moiety is connected to -L¹- inthe CNP prodrug of formula (IIe), (IIe-i) and (IIe-ii) through the aminefunctional group provided by the side chain of the lysine at position 9,if the CNP has the sequence of SEQ ID NO:24.

In another embodiment the CNP moiety is connected to -L- in the CNPprodrug of formula (IIe), (IIe-i) and (IIe-ii) through the aminefunctional group provided by the side chain of the lysine at position11, if the CNP has the sequence of SEQ ID NO:24.

In another embodiment the CNP moiety is connected to -L- in the CNPprodrug of formula (IIe), (IIe-i) and (IIe-ii) through the aminefunctional group provided by the side chain of the lysine at position15, if the CNP has the sequence of SEQ ID NO:24.

In another embodiment the CNP moiety is connected to -L- in the CNPprodrug of formula (IIe), (IIe-i) and (IIe-ii) through the aminefunctional group provided by the side chain of the lysine at position16, if the CNP has the sequence of SEQ ID NO:24.

In another embodiment the CNP moiety is connected to -L¹- in the CNPprodrug of formula (IIe), (IIe-i) and (IIe-ii) through the aminefunctional group provided by the side chain of the lysine at position20, if the CNP has the sequence of SEQ ID NO:24.

In a preferred embodiment said lysine side chain is part of the ringformed by the disulfide bridge between the cysteine residues atpositions 22 and 38, if the CNP moiety is of SEQ ID NO:24.

Accordingly, in one embodiment the CNP moiety is connected to -L¹- inthe CNP prodrug of formula (IIe), (IIe-i) and (IIe-ii) through the aminefunctional group provided by the side chain of the lysine at position26, if the CNP has the sequence of SEQ ID NO:24.

It is understood that the positions of the cysteines and lysinesmentioned above vary depending on the lengths of the CNP moiety and thatthe person skilled in the art will have no difficulty identifying thecorresponding cysteines and lysines in longer or shorter versions of theCNP moiety and also understands that for example some lysines may not bepresent in shorter CNP moieties. It is further understood that as aresult of for example site-directed mutagenesis there might be morelysine residues in the non-ring forming part and/or ring forming part ofthe CNP moiety.

In a preferred embodiment the CNP prodrug of the present invention is offormula (IIe), wherein c1 is about 450, -D is a CNP moiety having thesequence of SEQ ID NO:24 and is attached to -L¹- through the aminefunctional group provided by the side chain of the lysine at position26.

In another preferred embodiment the CNP prodrug of the present inventionis of formula (IIe-i), wherein c1 is about 450, the CNP moiety has thesequence of SEQ ID NO:24 and is attached to -L- through the aminefunctional group provided by the side chain of the lysine at position26.

In another preferred embodiment the CNP prodrug of the present inventionis of formula (IIe-ii), wherein c1 is about 450, the CNP moiety has thesequence of SEQ ID NO:24 and is attached to -L- through the aminefunctional group provided by the side chain of the lysine at position26.

In another preferred embodiment the CNP prodrug of the present inventionis of formula (IIe), wherein c1 is about 450, -D is a CNP moiety havingthe sequence of SEQ ID NO:20 and is attached to -L¹- through the aminefunctional group provided by the side chain of the lysine at position30.

In another preferred embodiment the CNP prodrug of the present inventionis of formula (IIe-i), wherein c1 is about 450, the CNP moiety has thesequence of SEQ ID NO:20 and is attached to -L¹- through the aminefunctional group provided by the side chain of the lysine at position30.

In another preferred embodiment the CNP prodrug of the present inventionis of formula (IIe-ii), wherein c1 is about 450, the CNP moiety has thesequence of SEQ ID NO:20 and is attached to -L- through the aminefunctional group provided by the side chain of the lysine at position30.

In a preferred embodiment the CNP prodrug of the present invention is offormula (IIe), wherein c1 is about 450, -D is a CNP moiety having thesequence of SEQ ID NO:21 and is attached to -L- through the aminefunctional group provided by the side chain of the lysine at position29.

In another preferred embodiment the CNP prodrug of the present inventionis of formula (IIe-i), wherein c1 is about 450, the CNP moiety has thesequence of SEQ ID NO:21 and is attached to -L- through the aminefunctional group provided by the side chain of the lysine at position29.

In another preferred embodiment the CNP prodrug of the present inventionis of formula (IIe-ii), wherein c1 is about 450, the CNP moiety has thesequence of SEQ ID NO:21 and is attached to -L- through the aminefunctional group provided by the side chain of the lysine at position29.

In a preferred embodiment the CNP prodrug of the present invention is offormula (IIe), wherein c1 is about 450, -D is a CNP moiety having thesequence of SEQ ID NO:22 and is attached to -L¹- through the aminefunctional group provided by the side chain of the lysine at position28.

In another preferred embodiment the CNP prodrug of the present inventionis of formula (IIe-i), wherein c1 is about 450, the CNP moiety has thesequence of SEQ ID NO:22 and is attached to -L¹- through the aminefunctional group provided by the side chain of the lysine at position28.

In another preferred embodiment the CNP prodrug of the present inventionis of formula (IIe-ii), wherein c1 is about 450, the CNP moiety has thesequence of SEQ ID NO:22 and is attached to -L¹- through the aminefunctional group provided by the side chain of the lysine at position28.

In a preferred embodiment the CNP prodrug of the present invention is offormula (IIe), wherein c1 is about 450, -D is a CNP moiety having thesequence of SEQ ID NO:23 and is attached to -L- through the aminefunctional group provided by the side chain of the lysine at position27.

In another preferred embodiment the CNP prodrug of the present inventionis of formula (IIe-i), wherein c1 is about 450, the CNP moiety has thesequence of SEQ ID NO:23 and is attached to -L- through the aminefunctional group provided by the side chain of the lysine at position27.

In another preferred embodiment the CNP prodrug of the present inventionis of formula (IIe-ii), wherein c1 is about 450, the CNP moiety has thesequence of SEQ ID NO:23 and is attached to -L- through the aminefunctional group provided by the side chain of the lysine at position27.

In a preferred embodiment the CNP prodrug of the present invention is offormula (IIe), wherein c1 is about 450, -D is a CNP moiety having thesequence of SEQ ID NO:30 and is attached to -L- through the aminefunctional group provided by the side chain of the lysine at position27.

In another preferred embodiment the CNP prodrug of the present inventionis of formula (IIe-i), wherein c1 is about 450, the CNP moiety has thesequence of SEQ ID NO:30 and is attached to -L¹- through the aminefunctional group provided by the side chain of the lysine at position27.

In another preferred embodiment the CNP prodrug of the present inventionis of formula (IIe-ii), wherein c1 is about 450, the CNP moiety has thesequence of SEQ ID NO:30 and is attached to -L¹- through the aminefunctional group provided by the side chain of the lysine at position27.

Accordingly, in a preferred embodiment the CNP agonist CNP prodrug ofthe present invention is of formula (IIe′)

-   -   wherein    -   the unmarked dashed line indicates the attachment to a nitrogen        provided by the side chain of the lysine at position 26 of the        CNP moiety of SEQ ID NO:24 by forming an amide bond; and    -   the dashed line marked with the asterisk indicates attachment to        a moiety

-   -   wherein    -   each c1 is an integer independently ranging from 400 to 500.

Preferably, each c1 of formula (IIe′) is about 450.

In another preferred embodiment the CNP prodrug of the present inventionis of formula (IIe-i′)

-   -   wherein    -   the unmarked dashed line indicates the attachment to a nitrogen        provided by the side chain of the lysine at position 26 of the        CNP moiety of SEQ ID NO:24 by forming an amide bond; and    -   the dashed line marked with the asterisk indicates attachment to        a moiety

-   -   wherein    -   each c1 is an integer independently ranging from 400 to 500.

Preferably, each c1 of formula (IIe-i′) is about 450.

In another preferred embodiment the CNP prodrug of the present inventionis of formula (IIe-ii′)

-   -   wherein    -   the unmarked dashed line indicates the attachment to a nitrogen        provided by the side chain of the lysine at position 26 of the        CNP moiety of SEQ ID NO:24 by forming an amide bond; and    -   the dashed line marked with the asterisk indicates attachment to        a moiety

-   -   wherein    -   each c1 is an integer independently ranging from 400 to 500.

Preferably, each c1 of formula (IIe-ii′) is about 450.

In another preferred embodiment the CNP agonist prodrug of the presentinvention is of formula (IIf)

-   -   wherein    -   the unmarked dashed line indicates the attachment to a nitrogen        of -D which is a CNP agonist moiety by forming an amide bond;        and the dashed line marked with the asterisk indicates        attachment to —Z having the structure

-   -   wherein    -   each —Z^(a) is

-   -   wherein    -   each c1 is an integer independently ranging from 200 to 250.

Preferably, each c1 of formula (IIf) is about 225.

In another preferred embodiment the CNP agonist prodrug of the presentinvention is of formula (IIf-i)

-   -   wherein    -   the unmarked dashed line indicates the attachment to a nitrogen        of -D which is a CNP agonist moiety by forming an amide bond;        and    -   the dashed line marked with the asterisk indicates attachment to        —Z having the structure

-   -   -   wherein        -   each —Z^(a) is

-   -   -   -   wherein            -   each c1 is an integer independently ranging from 200 to                250.

Preferably, each c1 of formula (IIf-i) is about 225.

In another preferred embodiment the CNP agonist prodrug of the presentinvention is of formula (IIf-ii)

-   -   wherein    -   the unmarked dashed line indicates the attachment to a nitrogen        of -D which is a CNP agonist moiety by forming an amide bond;        and    -   the dashed line marked with the asterisk indicates attachment to        —Z having the structure

-   -   -   wherein        -   each —Z^(a) is

-   -   -   -   wherein            -   each c1 is an integer independently ranging from 200 to                250.

Preferably, each c1 of formula (IIf-ii) is about 225.

Preferably -D of formula (IIf), (IIf-i) and (IIf-ii) is a CNP moiety,i.e. the prodrug of formula (IIf), (IIf-i) and (IIf-ii) is a CNPprodrug. Even more preferably -D of formula (IIf), (IIf-i) and (IIf-ii)is a CNP moiety having the sequence of SEQ ID NO:24, SEQ ID NO:25 or SEQID NO:30. Most preferably -D of formula (IIf), (IIf-i) and (IIf-ii) is aCNP moiety CNP having the sequence of SEQ ID NO:24. It is also preferredthat -D of formula (IIf), (IIf-i) and (IIf-ii) is a CNP moiety CNPhaving the sequence of SEQ ID NO:20. -D of formula (IIf), (IIf-i) and(IIf-ii) is a CNP moiety CNP having the sequence of SEQ ID NO:21. -D offormula (IIf), (IIf-i) and (IIf-ii) is a CNP moiety CNP having thesequence of SEQ ID NO:22. -D of formula (IIf), (IIf-i) and (IIf-ii) is aCNP moiety CNP having the sequence of SEQ ID NO:23. -D of formula (IIf),(IIf-i) and (IIf-ii) is a CNP moiety CNP having the sequence of SEQ IDNO:30.

In one embodiment -D of formula (IIf), (IIf-i) and (IIf-ii) is a CNPmoiety which is attached to -L¹- through the nitrogen of the N-terminalamine functional group of CNP.

In a preferred embodiment -D of formula (IIf), (IIf-i) and (IIf-ii) is aCNP moiety which is attached to -L¹- through a nitrogen provided by theamine functional group of a lysine side chain of the CNP moiety.

In one embodiment said lysine side chain is not part of the ring formedby the disulfide bridge between the cysteine residues at positions 22and 38, if the CNP moiety is of SEQ ID NO:24.

Accordingly, in one embodiment the CNP moiety is connected to -L- in theCNP prodrug of formula (IIf), (IIf-i) and (IIf-ii) through the aminefunctional group provided by the side chain of the lysine at position 9,if the CNP has the sequence of SEQ ID NO:24.

In another embodiment the CNP moiety is connected to -L- in the CNPprodrug of formula (IIf), (IIf-i) and (IIf-ii) through the aminefunctional group provided by the side chain of the lysine at position11, if the CNP has the sequence of SEQ ID NO:24.

In another embodiment the CNP moiety is connected to -L- in the CNPprodrug of formula (IIf), (IIf-i) and (IIf-ii) through the aminefunctional group provided by the side chain of the lysine at position15, if the CNP has the sequence of SEQ ID NO:24.

In another embodiment the CNP moiety is connected to -L- in the CNPprodrug of formula (IIf), (IIf-i) and (IIf-ii) through the aminefunctional group provided by the side chain of the lysine at position16, if the CNP has the sequence of SEQ ID NO:24.

In another embodiment the CNP moiety is connected to -L¹- in the CNPprodrug of formula (IIf), (IIf-i) and (IIf-ii) through the aminefunctional group provided by the side chain of the lysine at position20, if the CNP has the sequence of SEQ ID NO:24.

In a preferred embodiment said lysine side chain is part of the ringformed by the disulfide bridge between the cysteine residues atpositions 22 and 38, if the CNP moiety is of SEQ ID NO:24.

Accordingly, in one embodiment the CNP moiety is connected to -L¹- inthe CNP prodrug of formula (IIf), (IIf-i) and (IIf-ii) through the aminefunctional group provided by the side chain of the lysine at position26, if the CNP has the sequence of SEQ ID NO:24.

It is understood that the positions of the cysteines and lysinesmentioned above vary depending on the lengths of the CNP moiety and thatthe person skilled in the art will have no difficulty identifying thecorresponding cysteines and lysines in longer or shorter versions of theCNP moiety and also understands that for example some lysines may not bepresent in shorter CNP moieties. It is further understood that as aresult of for example site-directed mutagenesis there might be morelysine residues in the non-ring forming part and/or ring forming part ofthe CNP moiety.

In a preferred embodiment the CNP prodrug of the present invention is offormula (IIf), wherein c1 is about 225, -D is a CNP moiety having thesequence of SEQ ID NO:24 and is attached to -L- through the aminefunctional group provided by the side chain of the lysine at position26.

In another preferred embodiment the CNP prodrug of the present inventionis of formula (IIf-i), wherein c1 is about 225, the CNP moiety has thesequence of SEQ ID NO:24 and is attached to -L- through the aminefunctional group provided by the side chain of the lysine at position26.

In another preferred embodiment the CNP prodrug of the present inventionis of formula (IIf-ii), wherein c1 is about 225, the CNP moiety has thesequence of SEQ ID NO:24 and is attached to -L¹- through the aminefunctional group provided by the side chain of the lysine at position26.

In a preferred embodiment the CNP prodrug of the present invention is offormula (IIf), wherein c1 is about 225, -D is a CNP moiety having thesequence of SEQ ID NO:20 and is attached to -L¹- through the aminefunctional group provided by the side chain of the lysine at position30.

In another preferred embodiment the CNP prodrug of the present inventionis of formula (IIf-i), wherein c1 is about 225, the CNP moiety has thesequence of SEQ ID NO:20 and is attached to -L- through the aminefunctional group provided by the side chain of the lysine at position30.

In another preferred embodiment the CNP prodrug of the present inventionis of formula (IIf-ii), wherein c1 is about 225, the CNP moiety has thesequence of SEQ ID NO:20 and is attached to -L- through the aminefunctional group provided by the side chain of the lysine at position30.

In a preferred embodiment the CNP prodrug of the present invention is offormula (IIf), wherein c1 is about 225, -D is a CNP moiety having thesequence of SEQ ID NO:21 and is attached to -L- through the aminefunctional group provided by the side chain of the lysine at position29.

In another preferred embodiment the CNP prodrug of the present inventionis of formula (IIf-i), wherein c1 is about 225, the CNP moiety has thesequence of SEQ ID NO:21 and is attached to -L- through the aminefunctional group provided by the side chain of the lysine at position29.

In another preferred embodiment the CNP prodrug of the present inventionis of formula (IIf-ii), wherein c1 is about 225, the CNP moiety has thesequence of SEQ ID NO:21 and is attached to -L¹- through the aminefunctional group provided by the side chain of the lysine at position29.

In a preferred embodiment the CNP prodrug of the present invention is offormula (IIf), wherein c1 is about 225, -D is a CNP moiety having thesequence of SEQ ID NO:22 and is attached to -L¹- through the aminefunctional group provided by the side chain of the lysine at position28.

In another preferred embodiment the CNP prodrug of the present inventionis of formula (IIf-i), wherein c1 is about 225, the CNP moiety has thesequence of SEQ ID NO:22 and is attached to -L- through the aminefunctional group provided by the side chain of the lysine at position28.

In another preferred embodiment the CNP prodrug of the present inventionis of formula (IIf-ii), wherein c1 is about 225, the CNP moiety has thesequence of SEQ ID NO:22 and is attached to -L- through the aminefunctional group provided by the side chain of the lysine at position28.

In a preferred embodiment the CNP prodrug of the present invention is offormula (IIf), wherein c1 is about 225, -D is a CNP moiety having thesequence of SEQ ID NO:23 and is attached to -L- through the aminefunctional group provided by the side chain of the lysine at position27.

In another preferred embodiment the CNP prodrug of the present inventionis of formula (IIf-i), wherein c1 is about 225, the CNP moiety has thesequence of SEQ ID NO:23 and is attached to -L- through the aminefunctional group provided by the side chain of the lysine at position27.

In another preferred embodiment the CNP prodrug of the present inventionis of formula (IIf-ii), wherein c1 is about 225, the CNP moiety has thesequence of SEQ ID NO:23 and is attached to -L- through the aminefunctional group provided by the side chain of the lysine at position27.

In a preferred embodiment the CNP prodrug of the present invention is offormula (IIf), wherein c1 is about 225, -D is a CNP moiety having thesequence of SEQ ID NO:30 and is attached to -L¹- through the aminefunctional group provided by the side chain of the lysine at position27.

In another preferred embodiment the CNP prodrug of the present inventionis of formula (IIf-i), wherein c1 is about 225, the CNP moiety has thesequence of SEQ ID NO:30 and is attached to -L¹- through the aminefunctional group provided by the side chain of the lysine at position27.

In another preferred embodiment the CNP prodrug of the present inventionis of formula (IIf-ii), wherein c1 is about 225, the CNP moiety has thesequence of SEQ ID NO:30 and is attached to -L- through the aminefunctional group provided by the side chain of the lysine at position27.

In another preferred embodiment the CNP prodrug of the present inventionis of formula (IIf′)

-   -   wherein    -   the unmarked dashed line indicates the attachment to a nitrogen        provided by the side chain of the lysine at position 26 of the        CNP moiety of SEQ ID NO:24 by forming an amide bond; and    -   the dashed line marked with the asterisk indicates attachment to        —Z having the structure

-   -   -   wherein        -   each Z^(a) is

-   -   -   -   wherein            -   each c1 is an integer independently ranging from 200 to                250.

Preferably, each c1 of formula (IIf′) is about 225.

In another preferred embodiment the CNP prodrug of the present inventionis of formula (IIf-i′)

-   -   wherein    -   the unmarked dashed line indicates the attachment to a nitrogen        provided by the side chain of the lysine at position 26 of the        CNP moiety of SEQ ID NO:24 by forming an amide bond; and    -   the dashed line marked with the asterisk indicates attachment to        —Z having the structure

-   -   -   wherein        -   each Z^(a) is

-   -   -   -   wherein            -   each c1 is an integer independently ranging from 200 to                250.

Preferably, each c1 of formula (IIf-i′) is about 225.

In another preferred embodiment the CNP prodrug of the present inventionis of formula (IIf-ii′)

-   -   wherein    -   the unmarked dashed line indicates the attachment to a nitrogen        provided by the side chain of the lysine at position 26 of the        CNP moiety of SEQ ID NO:24 by forming an amide bond; and    -   the dashed line marked with the asterisk indicates attachment to        —Z having the structure

-   -   -   wherein        -   each Z^(a) is

-   -   -   -   wherein            -   each c1 is an integer independently ranging from 200 to                250.

Preferably, each c1 of formula (IIf-ii′) is about 225.

In an equally preferred embodiment the CNP agonist prodrug of thepresent invention is of formula (IIea)

-   -   wherein    -   the unmarked dashed line indicates the attachment to a nitrogen        of -D which is a CNP agonist moiety by forming an amide bond;    -   k is selected from the group consisting of 1, 2, 3, 4, 5, 6, 7,        8, 9, 10, 11 and 12; and the dashed line marked with the        asterisk indicates attachment to a moiety

-   -   -   wherein        -   each c1 is an integer independently ranging from 400 to 500.

Preferably, c1 of formula (IIea) is about 450.

Preferably, k of formula (IIea) is selected from the group consisting of2, 3, 4, 5, 6 and 7.

In an equally preferred embodiment the CNP agonist prodrug of thepresent invention is of formula (IIea-i)

-   -   wherein    -   the unmarked dashed line indicates the attachment to a nitrogen        of -D which is a CNP agonist moiety by forming an amide bond;    -   k is selected from the group consisting of 1, 2, 3, 4, 5, 6, 7,        8, 9, 10, 11 and 12; and    -   the dashed line marked with the asterisk indicates attachment to        a moiety

-   -   -   wherein        -   each c1 is an integer independently ranging from 400 to 500.

Preferably, k of formula (IIea-i) is selected from the group consistingof 2, 3, 4, 5, 6 and 7.

Preferably, c1 of formula (IIea-i) is about 450.

In another equally preferred embodiment the CNP agonist prodrug of thepresent invention is of formula (IIea-ii)

-   -   wherein    -   the unmarked dashed line indicates the attachment to a nitrogen        of -D which is a CNP agonist moiety by forming an amide bond;    -   k is selected from the group consisting of 1, 2, 3, 4, 5, 6, 7,        8, 9, 10, 11 and 12; and    -   the dashed line marked with the asterisk indicates attachment to        a moiety

-   -   -   wherein        -   each c1 is an integer independently ranging from 400 to 500.

Preferably, k of formula (IIea-ii) is selected from the group consistingof 2, 3, 4, 5, 6 and 7.

Preferably, c1 of formula (IIea-ii) is about 450.

Preferably -D of formula (IIea), (IIea-i) and (IIea-ii) is a CNP moiety,i.e. the prodrug of formula (IIea), (IIea-i) and (IIea-ii) is a CNPprodrug. Even more preferably -D of formula (IIea), (IIea-i) and(IIea-ii) is a CNP moiety having the sequence of SEQ ID NO:24, SEQ IDNO:25 or SEQ ID NO:30. Most preferably -D of formula (IIea), (IIea-i)and (IIea-ii) is a CNP moiety CNP having the sequence of SEQ ID NO:24.It is also preferred that -D of formula (IIea), (IIea-i) and (IIea-ii)is a CNP moiety CNP having the sequence of SEQ ID NO:20. It is alsopreferred that -D of formula (IIea), (IIea-i) and (IIea-ii) is a CNPmoiety CNP having the sequence of SEQ ID NO:21. It is also preferredthat -D of formula (IIea), (IIea-i) and (IIea-ii) is a CNP moiety CNPhaving the sequence of SEQ ID NO:22. It is also preferred that -D offormula (IIea), (IIea-i) and (IIea-ii) is a CNP moiety CNP having thesequence of SEQ ID NO:23. It is also preferred that -D of formula(IIea), (IIea-i) and (IIea-ii) is a CNP moiety CNP having the sequenceof SEQ ID NO:30.

In one embodiment -D of formula (IIea), (IIea-i) and (IIea-ii) is a CNPmoiety which is attached to -L¹- through the nitrogen of the N-terminalamine functional group of CNP.

In a preferred embodiment -D of formula (IIea), (IIea-i) and (IIea-ii)is a CNP moiety which is attached to -L¹- through a nitrogen provided bythe amine functional group of a lysine side chain of the CNP moiety.

In one embodiment said lysine side chain is not part of the ring formedby the disulfide bridge between the cysteine residues at positions 22and 38, if the CNP moiety is of SEQ ID NO:24.

Accordingly, in one embodiment the CNP moiety is connected to -L- in theCNP prodrug of formula (IIea), (IIea-i) and (IIea-ii) through the aminefunctional group provided by the side chain of the lysine at position 9,if the CNP has the sequence of SEQ ID NO:24.

In another embodiment the CNP moiety is connected to -L- in the CNPprodrug of formula (IIea), (IIea-i) and (IIea-ii) through the aminefunctional group provided by the side chain of the lysine at position11, if the CNP has the sequence of SEQ ID NO:24.

In another embodiment the CNP moiety is connected to -L¹- in the CNPprodrug of formula (IIea), (IIea-i) and (IIea-ii) through the aminefunctional group provided by the side chain of the lysine at position15, if the CNP has the sequence of SEQ ID NO:24.

In another embodiment the CNP moiety is connected to -L¹- in the CNPprodrug of formula (IIea), (IIea-i) and (IIea-ii) through the aminefunctional group provided by the side chain of the lysine at position16, if the CNP has the sequence of SEQ ID NO:24.

In another embodiment the CNP moiety is connected to -L¹- in the CNPprodrug of formula (IIea), (IIea-i) and (IIea-ii) through the aminefunctional group provided by the side chain of the lysine at position20, if the CNP has the sequence of SEQ ID NO:24.

In a preferred embodiment said lysine side chain is part of the ringformed by the disulfide bridge between the cysteine residues atpositions 22 and 38, if the CNP moiety is of SEQ ID NO:24.

Accordingly, in one embodiment the CNP moiety is connected to -L¹- inthe CNP prodrug of formula (IIea), (IIea-i) and (IIea-ii) through theamine functional group provided by the side chain of the lysine atposition 26, if the CNP has the sequence of SEQ ID NO:24.

It is understood that the positions of the cysteines and lysinesmentioned above vary depending on the lengths of the CNP moiety and thatthe person skilled in the art will have no difficulty identifying thecorresponding cysteines and lysines in longer or shorter versions of theCNP moiety and also understands that for example some lysines may not bepresent in shorter CNP moieties. It is further understood that as aresult of for example site-directed mutagenesis there might be morelysine residues in the non-ring forming part and/or ring forming part ofthe CNP moiety.

In a preferred embodiment the CNP prodrug of the present invention is offormula (IIea), wherein c1 is about 450, -D is a CNP moiety having thesequence of SEQ ID NO:24 and is attached to -L¹- through the aminefunctional group provided by the side chain of the lysine at position26.

In another preferred embodiment the CNP prodrug of the present inventionis of formula (IIea-i), wherein c1 is about 450, the CNP moiety has thesequence of SEQ ID NO:24 and is attached to -L- through the aminefunctional group provided by the side chain of the lysine at position26.

In another preferred embodiment the CNP prodrug of the present inventionis of formula (IIea-ii), wherein c1 is about 450, the CNP moiety has thesequence of SEQ ID NO:24 and is attached to -L- through the aminefunctional group provided by the side chain of the lysine at position26.

In a preferred embodiment the CNP prodrug of the present invention is offormula (IIea), wherein c1 is about 450, -D is a CNP moiety having thesequence of SEQ ID NO:20 and is attached to -L- through the aminefunctional group provided by the side chain of the lysine at position30.

In another preferred embodiment the CNP prodrug of the present inventionis of formula (IIea-i), wherein c1 is about 450, the CNP moiety has thesequence of SEQ ID NO:20 and is attached to -L- through the aminefunctional group provided by the side chain of the lysine at position30.

In another preferred embodiment the CNP prodrug of the present inventionis of formula (IIea-ii), wherein c1 is about 450, the CNP moiety has thesequence of SEQ ID NO:20 and is attached to -L- through the aminefunctional group provided by the side chain of the lysine at position30.

In a preferred embodiment the CNP prodrug of the present invention is offormula (IIea), wherein c1 is about 450, -D is a CNP moiety having thesequence of SEQ ID NO:21 and is attached to -L- through the aminefunctional group provided by the side chain of the lysine at position29.

In another preferred embodiment the CNP prodrug of the present inventionis of formula (IIea-i), wherein c1 is about 450, the CNP moiety has thesequence of SEQ ID NO:21 and is attached to -L¹- through the aminefunctional group provided by the side chain of the lysine at position29.

In another preferred embodiment the CNP prodrug of the present inventionis of formula (IIea-ii), wherein c1 is about 450, the CNP moiety has thesequence of SEQ ID NO:21 and is attached to -L¹- through the aminefunctional group provided by the side chain of the lysine at position29.

In a preferred embodiment the CNP prodrug of the present invention is offormula (IIea), wherein c1 is about 450, -D is a CNP moiety having thesequence of SEQ ID NO:22 and is attached to -L- through the aminefunctional group provided by the side chain of the lysine at position28.

In another preferred embodiment the CNP prodrug of the present inventionis of formula (IIea-i), wherein c1 is about 450, the CNP moiety has thesequence of SEQ ID NO:22 and is attached to -L- through the aminefunctional group provided by the side chain of the lysine at position28.

In another preferred embodiment the CNP prodrug of the present inventionis of formula (IIea-ii), wherein c1 is about 450, the CNP moiety has thesequence of SEQ ID NO:22 and is attached to -L- through the aminefunctional group provided by the side chain of the lysine at position28.

In a preferred embodiment the CNP prodrug of the present invention is offormula (IIea), wherein c1 is about 450, -D is a CNP moiety having thesequence of SEQ ID NO:23 and is attached to -L- through the aminefunctional group provided by the side chain of the lysine at position27.

In another preferred embodiment the CNP prodrug of the present inventionis of formula (IIea-i), wherein c1 is about 450, the CNP moiety has thesequence of SEQ ID NO:23 and is attached to -L¹- through the aminefunctional group provided by the side chain of the lysine at position27.

In another preferred embodiment the CNP prodrug of the present inventionis of formula (IIea-ii), wherein c1 is about 450, the CNP moiety has thesequence of SEQ ID NO:23 and is attached to -L¹- through the aminefunctional group provided by the side chain of the lysine at position27.

In a preferred embodiment the CNP prodrug of the present invention is offormula (IIea), wherein c1 is about 450, -D is a CNP moiety having thesequence of SEQ ID NO:30 and is attached to -L- through the aminefunctional group provided by the side chain of the lysine at position27.

In another preferred embodiment the CNP prodrug of the present inventionis of formula (IIea-i), wherein c1 is about 450, the CNP moiety has thesequence of SEQ ID NO:30 and is attached to -L- through the aminefunctional group provided by the side chain of the lysine at position27.

In another preferred embodiment the CNP prodrug of the present inventionis of formula (IIea-ii), wherein c1 is about 450, the CNP moiety has thesequence of SEQ ID NO:30 and is attached to -L- through the aminefunctional group provided by the side chain of the lysine at position27.

Accordingly, in a preferred embodiment the CNP prodrug of the presentinvention is of formula (IIea′)

-   -   wherein    -   the unmarked dashed line indicates the attachment to the        nitrogen provided by the side chain of the lysine at position 26        of the CNP moiety of SEQ ID NO:24 by forming an amide bond;    -   k is selected from the group consisting of 1, 2, 3, 4, 5, 6, 7,        8, 9, 10, 11 and 12; and    -   the dashed line marked with the asterisk indicates attachment to        a moiety

-   -   wherein    -   each c1 is an integer independently ranging from 400 to 500.

Preferably, k of formula (IIea′) is selected from the group consistingof 2, 3, 4, 5, 6 and 7.

Preferably, each c1 of formula (IIea′) is about 450.

In another preferred embodiment the CNP prodrug of the present inventionis of formula (IIea-i′)

-   -   wherein    -   the unmarked dashed line indicates the attachment to a nitrogen        provided by the side chain of the lysine at position 26 of the        CNP moiety of SEQ ID NO:24 by forming an amide bond;    -   k is selected from the group consisting of 1, 2, 3, 4, 5, 6, 7,        8, 9, 10, 11 and 12; and the dashed line marked with the        asterisk indicates attachment to a moiety

-   -   wherein    -   each c1 is an integer independently ranging from 400 to 500.

Preferably, k of formula (IIea-i′) is selected from the group consistingof 2, 3, 4, 5, 6 and 7.

Preferably, each c1 of formula (IIea-i′) is about 450.

In another preferred embodiment the CNP prodrug of the present inventionis of formula (Ilea-ii′)

-   -   wherein    -   the unmarked dashed line indicates the attachment to a nitrogen        provided by the side chain of the lysine at position 26 of the        CNP moiety of SEQ ID NO:24 by forming an amide bond;    -   k is selected from the group consisting of 1, 2, 3, 4, 5, 6, 7,        8, 9, 10, 11 and 12; and    -   the dashed line marked with the asterisk indicates attachment to        a moiety

-   -   wherein    -   each c1 is an integer independently ranging from 400 to 500.

Preferably, k of formula (IIea-ii′) is selected from the groupconsisting of 2, 3, 4, 5, 6 and 7.

Preferably, each c1 of formula (IIea-ii′) is about 450.

In another preferred embodiment the CNP prodrug of the present inventionis of formula (IIfa)

-   -   wherein    -   the unmarked dashed line indicates the attachment to a nitrogen        of -D which is a CNP moiety by forming an amide bond; and    -   the dashed line marked with the asterisk indicates attachment to        —Z having the structure

-   -   -   wherein        -   k is selected from the group consisting of 1, 2, 3, 4, 5, 6,            7, 8, 9, 10, 11 and 12;        -   each —Z^(a) is

-   -   -   -   wherein            -   each c1 is an integer independently ranging from 200 to                250.

Preferably, k of formula (IIfa) is selected from the group consisting of2, 3, 4, 5, 6 and 7.

Preferably, each c1 of formula (IIfa) is about 225.

In another preferred embodiment the CNP prodrug of the present inventionis of formula (IIfa-i)

-   -   wherein    -   the unmarked dashed line indicates the attachment to a nitrogen        of -D which is a CNP moiety by forming an amide bond; and    -   the dashed line marked with the asterisk indicates attachment to        —Z having the structure

-   -   -   wherein        -   k is selected from the group consisting of 1, 2, 3, 4, 5, 6,            7, 8, 9, 10, 11 and 12; each —Z^(a) is

-   -   -   -   wherein            -   each c1 is an integer independently ranging from 200 to                250.

Preferably, k of formula (IIfa-i) is selected from the group consistingof 2, 3, 4, 5, 6 and 7.

Preferably, each c1 of formula (IIfa-i) is about 225.

In another preferred embodiment the CNP prodrug of the present inventionis of formula (IIfa-ii)

-   -   wherein    -   the unmarked dashed line indicates the attachment to a nitrogen        of -D which is a CNP moiety by forming an amide bond; and    -   the dashed line marked with the asterisk indicates attachment to        —Z having the structure

-   -   -   wherein        -   k is selected from the group consisting of 1, 2, 3, 4, 5, 6,            7, 8, 9, 10, 11 and 12;        -   each —Z^(a) is

-   -   -   -   wherein            -   each c1 is an integer independently ranging from 200 to                250.

Preferably, each c1 of formula (IIfa-ii) is about 225.

In one embodiment the CNP moiety of the CNP prodrug of formula (IIfa),(IIfa-i) and (IIfa-ii) has the sequence of SEQ ID NO:25.

In another embodiment the CNP moiety of the CNP prodrug of formula(IIfa), (IIfa-i) and (IIfa-ii) has the sequence of SEQ ID NO:30.

In another embodiment the CNP moiety of the CNP prodrug of formula(IIfa), (IIfa-i) and (IIfa-ii) has the sequence of SEQ ID NO:20.

In another embodiment the CNP moiety of the CNP prodrug of formula(IIfa), (IIfa-i) and (IIfa-ii) has the sequence of SEQ ID NO:21.

In another embodiment the CNP moiety of the CNP prodrug of formula(IIfa), (IIfa-i) and (IIfa-ii) has the sequence of SEQ ID NO:22.

In another embodiment the CNP moiety of the CNP prodrug of formula(IIfa), (IIfa-i) and (IIfa-ii) has the sequence of SEQ ID NO:23.

In a preferred embodiment the CNP moiety of the CNP prodrug of formula(IIfa), (IIfa-i) and (IIfa-ii) has the sequence of SEQ ID NO:24.

In one embodiment the CNP moiety is attached to -L¹- in the CNP prodrugof formula (IIfa), (IIfa-i) and (IIfa-ii) through the nitrogen of theN-terminal amine functional group of CNP.

In another preferred embodiment the CNP prodrug of the present inventionis of formula (IIf a′)

-   -   wherein    -   the unmarked dashed line indicates the attachment to the        nitrogen provided by the side chain of the lysine at position 26        of the CNP moiety of SEQ ID NO:24 by forming an amide bond; and    -   the dashed line marked with the asterisk indicates attachment to        —Z having the structure

-   -   wherein    -   k is selected from the group consisting of 1, 2, 3, 4, 5, 6, 7,        8, 9, 10, 11 and 12;    -   each Z^(a) is

-   -   -   wherein        -   each c1 is an integer independently ranging from 200 to 250.

Preferably, k of formula (IIfa′) is selected from the group consistingof 2, 3, 4, 5, 6 and 7.

Preferably, each c1 of formula (IIfa′) is about 225.

In another preferred embodiment the CNP prodrug of the present inventionis of formula (IIfa-i′)

-   -   wherein    -   the unmarked dashed line indicates the attachment to a nitrogen        provided by the side chain of the lysine at position 26 of the        CNP moiety of SEQ ID NO:24 by forming an amide bond; and    -   the dashed line marked with the asterisk indicates attachment to        —Z having the structure

-   -   -   wherein        -   k is selected from the group consisting of 1, 2, 3, 4, 5, 6,            7, 8, 9, 10, 11 and 12;        -   each Z^(a) is

-   -   -   -   wherein            -   each c1 is an integer independently ranging from 200 to                250.

Preferably, k of formula (IIfa-i′) is selected from the group consistingof 2, 3, 4, 5, 6 and 7.

Preferably, each c1 of formula (IIfa-i′) is about 225.

In another preferred embodiment the CNP prodrug of the present inventionis of formula (IIfa-ii′)

-   -   wherein    -   the unmarked dashed line indicates the attachment to a nitrogen        provided by the side chain of the lysine at position 26 of the        CNP moiety of SEQ ID NO:24 by forming an amide bond; and    -   the dashed line marked with the asterisk indicates attachment to        —Z having the structure

-   -   -   wherein        -   k is selected from the group consisting of 1, 2, 3, 4, 5, 6,            7, 8, 9, 10, 11 and 12;        -   each Z^(a) is

-   -   -   -   wherein            -   each c1 is an integer independently ranging from 200 to                250.

Preferably, k of formula (IIfa-ii′) is selected from the groupconsisting of 2, 3, 4, 5, 6 and 7.

Preferably, each c1 of formula (IIfa-ii′) is about 225.

Another aspect of the present invention is a pharmaceutical compositioncomprising at least one controlled-release CNP agonist of the presentinvention and at least one excipient.

In one embodiment the pharmaceutical composition comprising at least onecontrolled-release CNP agonist of the present invention and at least oneexcipient is a liquid or suspension formulation. It is understood thatthe pharmaceutical composition is a suspension formulation if the atleast one controlled-release CNP agonist is water-insoluble.

In another embodiment the pharmaceutical composition comprising at leastone controlled-release CNP agonist of the present invention and at leastone excipient is a dry formulation.

Such liquid, suspension or dry pharmaceutical composition comprises atleast one excipient.

Excipients used in parenteral formulations may be categorized as, forexample, buffering agents, isotonicity modifiers, preservatives,stabilizers, anti-adsorption agents, oxidation protection agents,viscosifiers/viscosity enhancing agents, or other auxiliary agents.However, in some cases, one excipient may have dual or triple functions.Preferably, the at least one excipient comprised in the pharmaceuticalcomposition of the present invention is selected from the groupconsisting of

-   (i) Buffering agents: physiologically tolerated buffers to maintain    pH in a desired range, such as sodium phosphate, bicarbonate,    succinate, histidine, citrate and acetate, sulphate, nitrate,    chloride, pyruvate; antacids such as Mg(OH)₂ or ZnCO₃ may be also    used;-   (ii) Isotonicity modifiers: to minimize pain that can result from    cell damage due to osmotic pressure differences at the injection    depot; glycerin and sodium chloride are examples; effective    concentrations can be determined by osmometry using an assumed    osmolality of 285-315 mOsmol/kg for serum;-   (iii) Preservatives and/or antimicrobials: multidose parenteral    formulations require the addition of preservatives at a sufficient    concentration to minimize risk of patients becoming infected upon    injection and corresponding regulatory requirements have been    established; typical preservatives include m-cresol, phenol,    methylparaben, ethylparaben, propylparaben, butylparaben,    chlorobutanol, benzyl alcohol, phenylmercuric nitrate, thimerosol,    sorbic acid, potassium sorbate, benzoic acid, chlorocresol, and    benzalkonium chloride;-   (iv) Stabilizers: Stabilisation is achieved by strengthening of the    protein-stabilising forces, by destabilisation of the denatured    state, or by direct binding of excipients to the protein;    stabilizers may be amino acids such as alanine, arginine, aspartic    acid, glycine, histidine, lysine, proline, sugars such as glucose,    sucrose, trehalose, polyols such as glycerol, mannitol, sorbitol,    salts such as potassium phosphate, sodium sulphate, chelating agents    such as EDTA, hexaphosphate, ligands such as divalent metal ions    (zinc, calcium, etc.), other salts or organic molecules such as    phenolic derivatives; in addition, oligomers or polymers such as    cyclodextrins, dextran, dendrimers, PEG or PVP or protamine or HSA    may be used;-   (v) Anti-adsorption agents: Mainly ionic or non-ionic surfactants or    other proteins or soluble polymers are used to coat or adsorb    competitively to the inner surface of the formulation's container;    e.g., poloxamer (Pluronic F-68), PEG dodecyl ether (Brij 35),    polysorbate 20 and 80, dextran, polyethylene glycol,    PEG-polyhistidine, BSA and HSA and gelatins; chosen concentration    and type of excipient depends on the effect to be avoided but    typically a monolayer of surfactant is formed at the interface just    above the CMC value;-   (vi) Oxidation protection agents: antioxidants such as ascorbic    acid, ectoine, methionine, glutathione, monothioglycerol, morin,    polyethylenimine (PEI), propyl gallate, and vitamin E; chelating    agents such as citric acid, EDTA, hexaphosphate, and thioglycolic    acid may also be used;-   (vii) Viscosifiers or viscosity enhancers: retard settling of the    particles in the vial and syringe and are used in order to    facilitate mixing and resuspension of the particles and to make the    suspension easier to inject (i.e., low force on the syringe    plunger); suitable viscosifiers or viscosity enhancers are, for    example, carbomer viscosifiers like Carbopol 940, Carbopol Ultrez    10, cellulose derivatives like hydroxypropylmethylcellulose    (hypromellose, HPMC) or diethylaminoethyl cellulose (DEAE or    DEAE-C), colloidal magnesium silicate (Veegum) or sodium silicate,    hydroxyapatite gel, tricalcium phosphate gel, xanthans, carrageenans    like Satia gum UTC 30, aliphatic poly(hydroxy acids), such as    poly(D,L- or L-lactic acid) (PLA) and poly(glycolic acid) (PGA) and    their copolymers (PLGA), terpolymers of D,L-lactide, glycolide and    caprolactone, poloxamers, hydrophilic poly(oxyethylene) blocks and    hydrophobic poly(oxypropylene) blocks to make up a triblock of    poly(oxyethylene)-poly(oxypropylene)-poly(oxyethylene) (e.g.    Pluronic®), polyetherester copolymer, such as a polyethylene glycol    terephthalate/polybutylene terephthalate copolymer, sucrose acetate    isobutyrate (SAIB), dextran or derivatives thereof, combinations of    dextrans and PEG, polydimethylsiloxane, collagen, chitosan,    polyvinyl alcohol (PVA) and derivatives, polyalkylimides, poly    (acrylamide-co-diallyldimethyl ammonium (DADMA)),    polyvinylpyrrolidone (PVP), glycosaminoglycans (GAGs) such as    dermatan sulfate, chondroitin sulfate, keratan sulfate, heparin,    heparan sulfate, hyaluronan, ABA triblock or AB block copolymers    composed of hydrophobic A-blocks, such as polylactide (PLA) or    poly(lactide-co-glycolide) (PLGA), and hydrophilic B-blocks, such as    polyethylene glycol (PEG) or polyvinyl pyrrolidone; such block    copolymers as well as the abovementioned poloxamers may exhibit    reverse thermal gelation behavior (fluid state at room temperature    to facilitate administration and gel state above sol-gel transition    temperature at body temperature after injection);-   (viii) Spreading or diffusing agent: modifies the permeability of    connective tissue through the hydrolysis of components of the    extracellular matrix in the intrastitial space such as but not    limited to hyaluronic acid, a polysaccharide found in the    intercellular space of connective tissue; a spreading agent such as    but not limited to hyaluronidase temporarily decreases the viscosity    of the extracellular matrix and promotes diffusion of injected    drugs; and-   (ix) Other auxiliary agents: such as wetting agents, viscosity    modifiers, antibiotics, hyaluronidase; acids and bases such as    hydrochloric acid and sodium hydroxide are auxiliary agents    necessary for pH adjustment during manufacture.

Another aspect of the present invention is the controlled-release CNPagonist or a pharmaceutical composition comprising at least onecontrolled-release CNP agonist of the present invention for use as amedicament.

Preferably, said medicament is used in the treatment of a diseaseselected from the group consisting of achondroplasia, hypochondroplasia,short stature, dwarfism, osteochondrodysplasias, thanatophoricdysplasia, osteogenesis imperfecta, achondrogenesis, chondrodysplasiapunctata, homozygous achondroplasia, camptomelic dysplasia, congenitallethal hypophosphatasia, perinatal lethal type of osteogenesisimperfecta, short-rib polydactyly syndromes, rhizomelic type ofchondrodysplasia punctata, Jansen-type metaphyseal dysplasia,spondyloepiphyseal dysplasia congenita, atelosteogenesis, diastrophicdysplasia, congenital short femur, Langer-type mesomelic dysplasia,Nievergelt-type mesomelic dysplasia, Robinow syndrome, Reinhardtsyndrome, acrodysostosis, peripheral dysostosis, Kniest dysplasia,fibrochondrogenesis, Roberts syndrome, acromesomelic dysplasia,micromelia, Morquio syndrome, Kniest syndrome, metatrophic dysplasia,spondyloepimetaphyseal dysplasia, neurofibromatosis, Legius syndrome,LEOPARD syndrome, Noonan syndrome, hereditary gingival fibromatosis,neurofibromatosis type 1, Legius syndrome, cardiofaciocutaneoussyndrome, Costello syndrome, SHOX deficiency, idiopathic short stature,growth hormone deficiency, osteoarthritis, cleidocranial dysostosis,craniosynostosis (e.g., Muenke syndrome, Crouzon syndrome, Apertsyndrome, Jackson-Weiss syndrome, Pfeiffer syndrome, orCrouzonodermoskeletal syndrome), dactyly, brachydactyly, camptodactyly,polydactyly, syndactyly, dyssegmental dysplasia, enchondromatosis,fibrous dysplasia, hereditary multiple exostoses, hypophosphatemicrickets, Jaffe-Lichtenstein syndrome, Marfan syndrome, McCune-Albrightsyndrome, osteopetrosis and osteopoikilosis.

In another embodiment said medicament is used in the treatment of anophthalmic disorder, such as glaucoma and/or elevated intraocularpressure.

In another embodiment said medicament is used in the treatment of acancer disease associated with overactivation of FGFR3, e.g., multiplemyeloma, myeloproliferative syndrome, leukemia, plasma cell leukemia,lymphoma, glioblastoma, prostate cancer, bladder cancer, or mammarycancer.

In another embodiment said medicament is used in the treatment of avascular smooth muscle disorder, preferably selected from the groupconsisting of hypertension, restenosis, arteriosclerosis, acutedecompensated heart failure, congestive heart failure, cardiac edema,nephredema, hepatic edema, acute renal insufficiency, and chronic renalinsufficiency.

In another embodiment said medicament is used in the treatment ofhemorrhagic shock.

Preferably said medicament is used in the treatment of an achondroplasiaphenotype selected from the group consisting of growth retardation,skull deformities, orthodontic defects, cervical cord compression,spinal stenosis, hydrocephalus, hearing loss due to chronic otitis,cardiovascular disease, neurological disease, and obesity.

Most preferably said medicament is used in the treatment ofachondroplasia.

Another aspect of the present invention is the controlled-release CNPagonist or the pharmaceutical composition comprising at least onecontrolled-release CNP agonist of the present invention for use in amethod of treatment of a disease which can be treated with a CNPagonist.

Preferably, said disease is selected from the group consisting ofachondroplasia, hypochondroplasia, short stature, dwarfism,osteochondrodysplasias, thanatophoric dysplasia, osteogenesisimperfecta, achondrogenesis, chondrodysplasia punctata, homozygousachondroplasia, camptomelic dysplasia, congenital lethalhypophosphatasia, perinatal lethal type of osteogenesis imperfecta,short-rib polydactyly syndromes, rhizomelic type of chondrodysplasiapunctata, Jansen-type metaphyseal dysplasia, spondyloepiphysealdysplasia congenita, atelosteogenesis, diastrophic dysplasia, congenitalshort femur, Langer-type mesomelic dysplasia, Nievergelt-type mesomelicdysplasia, Robinow syndrome, Reinhardt syndrome, acrodysostosis,peripheral dysostosis, Kniest dysplasia, fibrochondrogenesis, Robertssyndrome, acromesomelic dysplasia, micromelia, Morquio syndrome, Kniestsyndrome, metatrophic dysplasia, spondyloepimetaphyseal dysplasia,neurofibromatosis, Legius syndrome, LEOPARD syndrome, Noonan syndrome,hereditary gingival fibromatosis, neurofibromatosis type 1, Legiussyndrome, cardiofaciocutaneous syndrome, Costello syndrome, SHOXdeficiency, idiopathic short stature, growth hormone deficiency,osteoarthritis, cleidocranial dysostosis, craniosynostosis (e.g., Muenkesyndrome, Crouzon syndrome, Apert syndrome, Jackson-Weiss syndrome,Pfeiffer syndrome, or Crouzonodermoskeletal syndrome), dactyly,brachydactyly, camptodactyly, polydactyly, syndactyly, dyssegmentaldysplasia, enchondromatosis, fibrous dysplasia, hereditary multipleexostoses, hypophosphatemic rickets, Jaffe-Lichtenstein syndrome, Marfansyndrome, McCune-Albright syndrome, osteopetrosis and osteopoikilosis.

In another embodiment the disease is an ophthalmic disorder, such asglaucoma and/or elevated intraocular pressure.

In another embodiment said disease is associated with overactivation ofFGFR3 in cancer, e.g., multiple myeloma, myeloproliferative syndrome,leukemia, plasma cell leukemia, lymphoma, glioblastoma, prostate cancer,bladder cancer, or mammary cancer.

In another embodiment said disease is a vascular smooth muscle disorder,preferably selected from the group consisting of hypertension,restenosis, arteriosclerosis, acute decompensated heart failure,congestive heart failure, cardiac edema, nephredema, hepatic edema,acute renal insufficiency, and chronic renal insufficiency.

In another embodiment said disease is hemorrhagic shock.

In another embodiment said disease is an achondroplasia phenotypeselected from the group consisting of growth retardation, skulldeformities, orthodontic defects, cervical cord compression, spinalstenosis, hydrocephalus, hearing loss due to chronic otitis,cardiovascular disease, neurological disease, and obesity.

Most preferably said disease is achondroplasia.

In one embodiment the patient undergoing the method of treatment of thepresent invention is a mammalian patient, preferably a human patient. Inone embodiment this human patient is an adult. In a preferred embodimentthe human patient is a pediatric patient.

Another aspect of the present invention is the use of thecontrolled-release CNP agonist or the pharmaceutical compositioncomprising at least one controlled-release CNP agonist of the presentinvention for the manufacture of a medicament for treating a diseasewhich can be treated with CNP.

Preferably, said disease is selected from the group consisting ofachondroplasia, hypochondroplasia, short stature, dwarfism,osteochondrodysplasias, thanatophoric dysplasia, osteogenesisimperfecta, achondrogenesis, chondrodysplasia punctata, homozygousachondroplasia, camptomelic dysplasia, congenital lethalhypophosphatasia, perinatal lethal type of osteogenesis imperfecta,short-rib polydactyly syndromes, rhizomelic type of chondrodysplasiapunctata, Jansen-type metaphyseal dysplasia, spondyloepiphysealdysplasia congenita, atelosteogenesis, diastrophic dysplasia, congenitalshort femur, Langer-type mesomelic dysplasia, Nievergelt-type mesomelicdysplasia, Robinow syndrome, Reinhardt syndrome, acrodysostosis,peripheral dysostosis, Kniest dysplasia, fibrochondrogenesis, Robertssyndrome, acromesomelic dysplasia, micromelia, Morquio syndrome, Kniestsyndrome, metatrophic dysplasia, spondyloepimetaphyseal dysplasia,neurofibromatosis, Legius syndrome, LEOPARD syndrome, Noonan syndrome,hereditary gingival fibromatosis, neurofibromatosis type 1, Legiussyndrome, cardiofaciocutaneous syndrome, Costello syndrome, SHOXdeficiency, idiopathic short stature, growth hormone deficiency,osteoarthritis, cleidocranial dysostosis, craniosynostosis (e.g., Muenkesyndrome, Crouzon syndrome, Apert syndrome, Jackson-Weiss syndrome,Pfeiffer syndrome, or Crouzonodermoskeletal syndrome), dactyly,brachydactyly, camptodactyly, polydactyly, syndactyly, dyssegmentaldysplasia, enchondromatosis, fibrous dysplasia, hereditary multipleexostoses, hypophosphatemic rickets, Jaffe-Lichtenstein syndrome, Marfansyndrome, McCune-Albright syndrome, osteopetrosis and osteopoikilosis.

In another embodiment said disease is an ophthalmic disorder, such asglaucoma and/or elevated intraocular pressure.

In another embodiment said disease is associated with overactivation ofFGFR3 in cancer, e.g., multiple myeloma, myeloproliferative syndrome,leukemia, plasma cell leukemia, lymphoma, glioblastoma, prostate cancer,bladder cancer, or mammary cancer.

In another embodiment said disease is a vascular smooth muscle disorder,preferably selected from the group consisting of hypertension,restenosis, arteriosclerosis, acute decompensated heart failure,congestive heart failure, cardiac edema, nephredema, hepatic edema,acute renal insufficiency, and chronic renal insufficiency.

In another embodiment said disease is hemorrhagic shock.

In another embodiment said disease is an achondroplasia phenotypeselected from the group consisting of growth retardation, skulldeformities, orthodontic defects, cervical cord compression, spinalstenosis, hydrocephalus, hearing loss due to chronic otitis,cardiovascular disease, neurological disease, and obesity.

Most preferably said disease is achondroplasia.

In one embodiment the disease to be treated with the controlled-releaseCNP agonist or the pharmaceutical composition comprising at least onecontrolled-release CNP agonist of the present invention occurs in amammalian patient, preferably in a human patient. In one embodiment thishuman patient is an adult. In a preferred embodiment the human patientis a pediatric patient.

A further aspect of the present invention is a method of treating,controlling, delaying or preventing in a mammalian patient, preferably ahuman patient, in need of the treatment of one or more diseases whichcan be treated with a CNP agonist, comprising the step of administeringto said patient in need thereof a therapeutically effective amount ofthe controlled-release CNP agonist or a pharmaceutical compositioncomprising at least one controlled-release CNP agonist of the presentinvention. In one embodiment the human patient is an adult. In apreferred embodiment the human patient is a pediatric patient.

Preferably, the one or more diseases which can be treated with CNP isselected from the group consisting of achondroplasia, hypochondroplasia,short stature, dwarfism, osteochondrodysplasias, thanatophoricdysplasia, osteogenesis imperfecta, achondrogenesis, chondrodysplasiapunctata, homozygous achondroplasia, camptomelic dysplasia, congenitallethal hypophosphatasia, perinatal lethal type of osteogenesisimperfecta, short-rib polydactyly syndromes, rhizomelic type ofchondrodysplasia punctata, Jansen-type metaphyseal dysplasia,spondyloepiphyseal dysplasia congenita, atelosteogenesis, diastrophicdysplasia, congenital short femur, Langer-type mesomelic dysplasia,Nievergelt-type mesomelic dysplasia, Robinow syndrome, Reinhardtsyndrome, acrodysostosis, peripheral dysostosis, Kniest dysplasia,fibrochondrogenesis, Roberts syndrome, acromesomelic dysplasia,micromelia, Morquio syndrome, Kniest syndrome, metatrophic dysplasia,spondyloepimetaphyseal dysplasia, neurofibromatosis, Legius syndrome,LEOPARD syndrome, Noonan syndrome, hereditary gingival fibromatosis,neurofibromatosis type 1, Legius syndrome, cardiofaciocutaneoussyndrome, Costello syndrome, SHOX deficiency, idiopathic short stature,growth hormone deficiency, osteoarthritis, cleidocranial dysostosis,craniosynostosis (e.g., Muenke syndrome, Crouzon syndrome, Apertsyndrome, Jackson-Weiss syndrome, Pfeiffer syndrome, orCrouzonodermoskeletal syndrome), dactyly, brachydactyly, camptodactyly,polydactyly, syndactyly, dyssegmental dysplasia, enchondromatosis,fibrous dysplasia, hereditary multiple exostoses, hypophosphatemicrickets, Jaffe-Lichtenstein syndrome, Marfan syndrome, McCune-Albrightsyndrome, osteopetrosis and osteopoikilosis.

In another embodiment the one or more diseases which can be treated withCNP is an ophthalmic disorder, such as glaucoma and/or elevatedintraocular pressure.

In another embodiment the one or more diseases which can be treated withCNP is associated with overactivation of FGFR3 in cancer, e.g., multiplemyeloma, myeloproliferative syndrome, leukemia, plasma cell leukemia,lymphoma, glioblastoma, prostate cancer, bladder cancer, or mammarycancer.

In another embodiment the one or more diseases which can be treated withCNP is a vascular smooth muscle disorder, preferably selected from thegroup consisting of hypertension, restenosis, arteriosclerosis, acutedecompensated heart failure, congestive heart failure, cardiac edema,nephredema, hepatic edema, acute renal insufficiency, and chronic renalinsufficiency.

In another embodiment the one or more disease which can be treated withCNP is hemorrhagic shock.

In another embodiment the one or more diseases which can be treated withCNP is an achondroplasia phenotype selected from the group consisting ofgrowth retardation, skull deformities, orthodontic defects, cervicalcord compression, spinal stenosis, hydrocephalus, hearing loss due tochronic otitis, cardiovascular disease, neurological disease, andobesity.

Most preferably the one or more diseases which can be treated with CNPis achondroplasia.

An additional aspect of the present invention is a method ofadministering the controlled-release CNP agonist or the pharmaceuticalcomposition comprising at least one controlled-release CNP agonist ofthe present invention, wherein the method comprises the step ofadministering the controlled-release CNP agonist or the pharmaceuticalcomposition comprising at least one controlled-release CNP agonist ofthe present invention via topical, enteral or parenteral administrationand by methods of external application, injection or infusion, includingintraarticular, periarticular, intradermal, subcutaneous, intramuscular,intravenous, intraosseous, intraperitoneal, intrathecal, intracapsular,intraorbital, intravitreal, intratympanic, intravesical, intracardiac,transtracheal, subcuticular, subcapsular, subarachnoid, intraspinal,intraventricular, intrasternal injection and infusion, direct deliveryto the brain via implanted device allowing delivery of the invention orthe like to brain tissue or brain fluids (e.g., Ommaya Reservoir),direct intracerebroventricular injection or infusion, injection orinfusion into brain or brain associated regions, injection into thesubchoroidal space, retro-orbital injection and ocular instillation,preferably via subcutaneous injection.

In a preferred embodiment, the present invention relates to acontrolled-release CNP agonist or a pharmaceutical compositioncomprising at least one controlled-release CNP agonist of the presentinvention, for use in the treatment of achondroplasia via subcutaneousinjection.

In a further aspect the present invention relates to a pharmaceuticalcomposition comprising at least one controlled-release CNP agonist ofthe present invention or a pharmaceutically acceptable salt thereof,wherein the pharmaceutical composition comprises at least one furtherbiologically active moiety or drug.

The at least one further biologically active moiety or drug may be inits free form (i.e. in the form of a free drug), may be in the form of astable conjugate or may be in the form of a controlled-release compound.

In one embodiment, the at least one further biologically active moietyor drug is a drug in its free form, i.e. the pharmaceutical compositionof the present invention comprises at least one controlled-release CNPagonist and at least one further drug.

Preferably, the at least one further drug is selected from the groupconsisting of antihistamins; human anti-FGFR3 antibodies; soluble formsof human fibroblast growth factor receptor 3; tyrosine kinaseinhibitors; statins; CNP agonists; growth hormone; IGF-1; ANP; BNP;inhibitors of peptidases and proteases; and inhibitors of NPR-C.

A preferred antihistamin is meclozine.

A preferred tyrosine kinase inhibitor is NVP-BGJ398.

A preferred statin is rosuvastatin.

A preferred CNP agonist for the at least one further drug is vosoritide.

Preferred inhibitors of peptidases and proteases are NEP and furininhibitors.

A preferred inhibitor for NEP are thiorphan and candoxatril.

Preferred inhibitors of NPR-C are the fragment of SEQ ID NO:98(FGIPMDRIGRNPR) and antibody B701.

Preferred inhibitors of tyrosine kinases are as disclosed in U.S. Pat.Nos. 6,329,375 and 6,344,459, which are herewith incorporated byreference.

In one embodiment the at least one further drug is an antihistamin.

In another embodiment the at least one further drug is a humananti-FGFR3 antibody.

In another embodiment the at least one further drug is a soluble formsof human fibroblast growth factor receptor 3 (sFGFR3).

In another embodiment the at least one further drug is a tyrosine kinaseinhibitor.

In another embodiment the at least one further drug is a statin.

In another embodiment the at least one further drug is a growth hormone.

In another embodiment the at least one further drug is a CNP agonist.

In another embodiment the at least one further drug is IGF-1.

In another embodiment the at least one further drug is ANP.

In another embodiment the at least one further is BNP.

In another embodiment the at least one further drug is an inhibitor ofpeptidases and proteases.

In another embodiment the at least one further drug is an inhibitor ofNPR-C.

In another embodiment, the at least one further biologically activemoiety or drug is in the form of a stable conjugate.

In one embodiment the at least one further biologically active moiety inthe form of a stable conjugate comprises at least one biologicallyactive moiety covalently conjugated through a stable linkage to apolymeric moiety, preferably to a water-soluble polymeric moiety, eitherdirectly or through a spacer moiety.

Preferably, such polymeric moiety, even more preferably water-solublepolymeric moiety, comprises a polymer selected from the group consistingof 2-methacryloyl-oxyethyl phosphoyl cholins, poly(acrylic acids),poly(acrylates), poly(acrylamides), poly(alkyloxy) polymers,poly(amides), poly(amidoamines), poly(amino acids), poly(anhydrides),poly(aspartamides), poly(butyric acids), poly(glycolic acids),polybutylene terephthalates, poly(caprolactones), poly(carbonates),poly(cyanoacrylates), poly(dimethylacrylamides), poly(esters),poly(ethylenes), poly(ethyleneglycols), poly(ethylene oxides),poly(ethyl phosphates), poly(ethyloxazolines), poly(glycolic acids),poly(hydroxyethyl acrylates), poly(hydroxyethyl-oxazolines),poly(hydroxymethacrylates), poly(hydroxypropylmethacrylamides),poly(hydroxypropyl methacrylates), poly(hydroxypropyloxazolines),poly(imino carbonates), poly(lactic acids), poly(lactic-co-glycolicacids), poly(methacrylamides), poly(methacrylates),poly(methyloxazolines), poly(organophosphazenes), poly(ortho esters),poly(oxazolines), poly(propylene glycols), poly(siloxanes),poly(urethanes), poly(vinyl alcohols), poly(vinyl amines),poly(vinylmethylethers), poly(vinylpyrrolidones), silicones, celluloses,carbomethyl celluloses, hydroxypropyl methylcelluloses, chitins,chitosans, dextrans, dextrins, gelatins, hyaluronic acids andderivatives, functionalized hyaluronic acids, mannans, pectins,rhamnogalacturonans, starches, hydroxyalkyl starches, hydroxyethylstarches and other carbohydrate-based polymers, xylans, and copolymersthereof.

In another embodiment the at least one further biologically activemoiety in the form of a stable conjugate is covalently conjugatedthrough a stable linkage to an albumin-binding moiety. Preferably, saidalbumin-binding moiety is a C₈₋₂₄ alkyl moiety or fatty acid derivative.Preferred fatty acid derivatives are those disclosed in WO 2005/027978A2 and WO 2014/060512 A1 which are herewith incorporated by reference.

Preferably, the at least one further biologically active moiety in theform of a stable conjugate comprises a biologically active moietyselected from the group consisting of antihistamins; human anti-FGFR3antibodies; soluble forms of human fibroblast growth factor receptor 3(sFGFR3); tyrosine kinase inhibitors; statins; CNP agonists; growthhormone; IGF-1; ANP; BNP; inhibitors of peptidases and proteases; andinhibitors of NPR-C.

A preferred antihistamin is meclozine.

A preferred tyrosine kinase inhibitor is NVP-BGJ398.

A preferred statin is rosuvastatin.

A preferred CNP agonist for the at least one further biologically activemoiety is vosoritide.

Preferred inhibitors of peptidases and proteases are NEP and furininhibitors.

A preferred inhibitor for NEP are thiorphan and candoxatril.

Preferred inhibitors of NPR-C are the fragment of SEQ ID NO:98(FGIPMDRIGRNPR) and antibody B701.

Preferred inhibitors of tyrosine kinases are as disclosed in U.S. Pat.Nos. 6,329,375 and 6,344,459, which are herewith incorporated byreference.

In one embodiment the at least one further biologically active moiety inthe form of a stable conjugate comprises an antihistamin moiety.

In another embodiment the at least one further biologically activemoiety in the form of a stable conjugate comprises a human anti-FGFR3antibody moiety.

In another embodiment the at least one further biologically activemoiety in the form of a stable conjugate comprises a soluble forms ofhuman fibroblast growth factor receptor 3 (sFGFR3) moiety.

In another embodiment the at least one further biologically activemoiety in the form of a stable conjugate comprises a tyrosine kinaseinhibitor moiety.

In another embodiment the at least one further biologically activemoiety in the form of a stable conjugate comprises a statin moiety.

In another embodiment the at least one further biologically activemoiety in the form of a stable conjugate comprises a growth hormonemoiety.

In another embodiment the at least one further biologically activemoiety in the form of a stable conjugate comprises a CNP agonist moiety.

In another embodiment the at least one further biologically activemoiety in the form of a stable conjugate comprises an IGF-1 moiety.

In another embodiment the at least one further biologically activemoiety in the form of a stable conjugate comprises an ANP moiety.

In another embodiment the at least one further biologically activemoiety in the form of a stable conjugate comprises a BNP moiety.

In another embodiment the at least one further biologically activemoiety in the form of a stable conjugate comprises an inhibitor ofpeptidases and proteases moiety.

In another embodiment the at least one further biologically activemoiety in the form of a stable conjugate comprises an inhibitor of NPR-Cmoiety.

In another embodiment the at least one further biologically activemoiety or drug is in the form of a controlled-release compound.

Preferably, the at least one further biologically active moiety or drugin the form of a controlled-release compound comprises at least onebiologically active moiety or drug selected from the group consisting ofantihistamins; human anti-FGFR3 antibodies; soluble forms of humanfibroblast growth factor receptor 3; statins; CNP agonists; growthhormone; IGF-1; ANP; BNP; inhibitors of peptidases and proteases;inhibitors of tyrosine kinases; and inhibitors of NPR-C.

A preferred antihistamin is meclozine.

A preferred tyrosine kinase inhibitor is NVP-BGJ398.

A preferred statin is rosuvastatin.

A preferred CNP agonist for the at least one further drug is vosoritide.

Preferred inhibitors of peptidases and proteases are NEP and furininhibitors.

A preferred inhibitor for NEP are thiorphan and candoxatril.

Preferred inhibitors of NPR-C are the fragment of SEQ ID NO:98(FGIPMDRIGRNPR) and antibody B701.

Preferred inhibitors of tyrosine kinases are as disclosed in U.S. Pat.Nos. 6,329,375 and 6,344,459, which are herewith incorporated byreference.

In one embodiment the at least one further biologically active moiety ordrug in the form of a controlled-release comprises an antihistaminmoiety or drug.

In another embodiment the at least one further biologically activemoiety or drug in the form of a controlled-release comprises a humananti-FGFR3 antibody moiety or drug.

In another embodiment the at least one further biologically activemoiety or drug in the form of a controlled-release comprises a solubleforms of human fibroblast growth factor receptor 3 (sFGFR3) moiety ordrug.

In another embodiment the at least one further biologically activemoiety or drug in the form of a controlled-release comprises a tyrosinekinase inhibitor moiety or drug.

In another embodiment the at least one further biologically activemoiety or drug in the form of a controlled-release comprises a statinmoiety or drug.

In another embodiment the at least one further biologically activemoiety or drug in the form of a controlled-release comprises a growthhormone moiety or drug.

In another embodiment the at least one further biologically activemoiety or drug in the form of a controlled-release comprises a CNPagonist moiety or drug.

In another embodiment the at least one further biologically activemoiety or drug in the form of a controlled-release comprises an IGF-1moiety or drug.

In another embodiment the at least one further biologically activemoiety or drug in the form of a controlled-release comprises an ANPmoiety or drug.

In another embodiment the at least one further biologically activemoiety or drug in the form of a controlled-release comprises a BNPmoiety or drug.

In another embodiment the at least one further biologically activemoiety or drug in the form of a controlled-release comprises aninhibitor of peptidases and proteases moiety or drug.

In another embodiment the at least one further biologically activemoiety or drug in the form of a controlled-release comprises aninhibitor of NPR-C moiety or drug.

In one embodiment the at least one further biologically active moiety ordrug in the form of a controlled-release compound is water-insoluble.

Preferably, such water-insoluble controlled-release compound is selectedfrom the group consisting of crystals, nanoparticles, microparticles,nanospheres and microspheres.

In one embodiment the at least one further biologically active moiety ordrug in the form of a water-insoluble controlled-release compound is acrystal comprising at least one drug or biologically active moiety.

In another embodiment the at least one further biologically activemoiety or drug in the form of a water-insoluble controlled-releasecompound is a nanoparticle comprising at least one drug or biologicallyactive moiety.

In another embodiment the at least one further biologically activemoiety or drug in the form of a water-insoluble controlled-releasecompound is a microparticle comprising at least one drug or biologicallyactive moiety.

In another embodiment the at least one further biologically activemoiety or drug in the form of a water-insoluble controlled-releasecompound is a nanosphere comprising at least one drug or biologicallyactive moiety.

In another embodiment the at least one further biologically activemoiety or drug in the form of a water-insoluble controlled-releasecompound is a microsphere comprising at least one drug or biologicallyactive moiety.

In one embodiment the at least one further biologically active moiety ordrug in the form of a water-insoluble controlled-release compound is avesicle comprising at least one drug or biologically active moiety.Preferably, such vesicle comprising at least one drug or biologicallyactive moiety is a micelle, liposome or polymersome.

In one embodiment the at least one further biologically active moiety ordrug in the form of a water-insoluble controlled-release compound is amicelle comprising at least one drug or biologically active moiety.

In another embodiment the at least one further biologically activemoiety or drug in the form of a water-insoluble controlled-releasecompound is a liposome comprising at least one drug or biologicallyactive moiety. Preferably, such liposome is selected from the groupconsisting of aquasomes; non-ionic surfactant vesicles, such as niosomesand proniosomes; cationic liposomes, such as LeciPlex; transfersomes;ethosomes; ufasomes; sphingosomes; and pharmacosomes.

In another embodiment the at least one further biologically activemoiety or drug in the form of a water-insoluble controlled-releasecompound is a polymersome at least one drug or biologically activemoiety.

In another embodiment the at least one further biologically activemoiety or drug in the form of a water-insoluble controlled-releasecompound comprises at least one biologically active moiety or drugnon-covalently embedded in a water-insoluble polymer. Preferably, suchwater-insoluble polymer comprises a polymer selected from the groupconsisting of 2-methacryloyl-oxyethyl phosphoyl cholins, poly(acrylicacids), poly(acrylates), poly(acrylamides), poly(alkyloxy) polymers,poly(amides), poly(amidoamines), poly(amino acids), poly(anhydrides),poly(aspartamides), poly(butyric acids), poly(glycolic acids),polybutylene terephthalates, poly(caprolactones), poly(carbonates),poly(cyanoacrylates), poly(dimethylacrylamides), poly(esters),poly(ethylenes), poly(ethyleneglycols), poly(ethylene oxides),poly(ethyl phosphates), poly(ethyloxazolines), poly(glycolic acids),poly(hydroxyethyl acrylates), poly(hydroxyethyl-oxazolines),poly(hydroxymethacrylates), poly(hydroxypropylmethacrylamides),poly(hydroxypropyl methacrylates), poly(hydroxypropyloxazolines),poly(imino carbonates), poly(lactic acids), poly(lactic-co-glycolicacids), poly(methacrylamides), poly(methacrylates),poly(methyloxazolines), poly(organophosphazenes), poly(ortho esters),poly(oxazolines), poly(propylene glycols), poly(siloxanes),poly(urethanes), poly(vinyl alcohols), poly(vinyl amines),poly(vinylmethylethers), poly(vinylpyrrolidones), silicones, celluloses,carbomethyl celluloses, hydroxypropyl methylcelluloses, chitins,chitosans, dextrans, dextrins, gelatins, hyaluronic acids andderivatives, functionalized hyaluronic acids, mannans, pectins,rhamnogalacturonans, starches, hydroxyalkyl starches, hydroxyethylstarches and other carbohydrate-based polymers, xylans, and copolymersthereof.

In a preferred embodiment the at least one further biologically activemoiety or drug in the form of a water-insoluble controlled-releasecompound comprises at least one drug or biologically active moietynon-covalently embedded in poly(lactic-co-glycolic acid) (PLGA).

In another embodiment the at least one further biologically activemoiety or drug in the form of a water-insoluble controlled-releasecompound comprises at least one biologically active moiety covalentlyand reversibly conjugated to a water-insoluble polymer. Preferably suchwater-insoluble polymer comprises a polymer selected from the groupconsisting of 2-methacryloyl-oxyethyl phosphoyl cholins, poly(acrylicacids), poly(acrylates), poly(acrylamides), poly(alkyloxy) polymers,poly(amides), poly(amidoamines), poly(amino acids), poly(anhydrides),poly(aspartamides), poly(butyric acids), poly(glycolic acids),polybutylene terephthalates, poly(caprolactones), poly(carbonates),poly(cyanoacrylates), poly(dimethylacrylamides), poly(esters),poly(ethylenes), poly(ethyleneglycols), poly(ethylene oxides),poly(ethyl phosphates), poly(ethyloxazolines), poly(glycolic acids),poly(hydroxyethyl acrylates), poly(hydroxyethyl-oxazolines),poly(hydroxymethacrylates), poly(hydroxypropylmethacrylamides),poly(hydroxypropyl methacrylates), poly(hydroxypropyloxazolines),poly(iminocarbonates), poly(lactic acids), poly(lactic-co-glycolicacids), poly(methacrylamides), poly(methacrylates),poly(methyloxazolines), poly(organophosphazenes), poly(ortho esters),poly(oxazolines), poly(propylene glycols), poly(siloxanes),poly(urethanes), poly(vinyl alcohols), poly(vinyl amines),poly(vinylmethylethers), poly(vinylpyrrolidones), silicones, celluloses,carbomethyl celluloses, hydroxypropyl methylcelluloses, chitins,chitosans, dextrans, dextrins, gelatins, hyaluronic acids andderivatives, functionalized hyaluronic acids, mannans, pectins,rhamnogalacturonans, starches, hydroxyalkyl starches, hydroxyethylstarches and other carbohydrate-based polymers, xylans, and copolymersthereof.

Preferably, the at least one further biologically active moiety or drugin the form of a water-insoluble controlled-release compound comprisesat least one biologically active moiety or drug selected from the groupconsisting of antihistamins; human anti-FGFR3 antibodies; soluble formsof human fibroblast growth factor receptor 3; tyrosine kinaseinhibitors; statins; CNP agonists; growth hormone; IGF-1; ANP; BNP;inhibitors of peptidases and proteases; and inhibitors of NPR-C.

A preferred antihistamin is meclozine.

A preferred tyrosine kinase inhibitor is NVP-BGJ398.

A preferred statin is rosuvastatin.

A preferred CNP agonist for the at least one further drug is vosoritide.

Preferred inhibitors of peptidases and proteases are NEP and furininhibitors.

A preferred inhibitor for NEP are thiorphan and candoxatril.

Preferred inhibitors of NPR-C are the fragment of SEQ ID NO:98(FGIPMDRIGRNPR) and antibody B701.

Preferred inhibitors of tyrosine kinases are as disclosed in U.S. Pat.Nos. 6,329,375 and 6,344,459, which are herewith incorporated byreference.

In one embodiment the at least one further biologically active moiety ordrug in the form of a water-insoluble controlled-release comprises anantihistamin moiety or drug.

In another embodiment the at least one further biologically activemoiety or drug in the form of a water-insoluble controlled-releasecomprises a human anti-FGFR3 antibody moiety or drug.

In another embodiment the at least one further biologically activemoiety or drug in the form of a water-insoluble controlled-releasecomprises a soluble forms of human fibroblast growth factor receptor 3(sFGFR3) moiety or drug.

In another embodiment the at least one further biologically activemoiety or drug in the form of a water-insoluble controlled-releasecomprises a tyrosine kinase inhibitor moiety or drug.

In another embodiment the at least one further biologically activemoiety or drug in the form of a water-insoluble controlled-releasecomprises a statin moiety or drug.

In another embodiment the at least one further biologically activemoiety or drug in the form of a water-insoluble controlled-releasecomprises a growth hormone moiety or drug.

In another embodiment the at least one further biologically activemoiety or drug in the form of a water-insoluble controlled-releasecomprises a CNP agonist moiety.

In another embodiment the at least one further biologically activemoiety or drug in the form of a water-insoluble controlled-releasecomprises an IGF-1 moiety or drug.

In another embodiment the at least one further biologically activemoiety or drug in the form of a water-insoluble controlled-releasecomprises an ANP moiety or drug.

In another embodiment the at least one further biologically activemoiety or drug in the form of a water-insoluble controlled-releasecomprises a BNP moiety or drug.

In another embodiment the at least one further biologically activemoiety or drug in the form of a water-insoluble controlled-releasecomprises an inhibitor of peptidases and proteases moiety or drug.

In another embodiment the at least one further biologically activemoiety or drug in the form of a water-insoluble controlled-releasecomprises an inhibitor of NPR-C moiety or drug.

In another embodiment the at least one further biologically activemoiety or drug in the form of a controlled-release compound iswater-soluble.

In one embodiment the at least one further biologically active moiety ordrug in the form of a water-soluble controlled-release compoundcomprises at least one biologically active moiety covalently conjugatedthrough a reversible linkage to a water-soluble polymeric moiety, eitherdirectly or through a spacer moiety.

Preferably, such water-soluble polymeric moiety comprises a polymerselected from the group consisting of 2-methacryloyl-oxyethyl phosphoylcholins, poly(acrylic acids), poly(acrylates), poly(acrylamides),poly(alkyloxy) polymers, poly(amides), poly(amidoamines), poly(aminoacids), poly(anhydrides), poly(aspartamides), poly(butyric acids),poly(glycolic acids), polybutylene terephthalates, poly(caprolactones),poly(carbonates), poly(cyanoacrylates), poly(dimethylacrylamides),poly(esters), poly(ethylenes), poly(ethyleneglycols), poly(ethyleneoxides), poly(ethyl phosphates), poly(ethyloxazolines), poly(glycolicacids), poly(hydroxyethyl acrylates), poly(hydroxyethyl-oxazolines),poly(hydroxymethacrylates), poly(hydroxypropylmethacrylamides),poly(hydroxypropyl methacrylates), poly(hydroxypropyloxazolines),poly(iminocarbonates), poly(lactic acids), poly(lactic-co-glycolicacids), poly(methacrylamides), poly(methacrylates),poly(methyloxazolines), poly(organophosphazenes), poly(ortho esters),poly(oxazolines), poly(propylene glycols), poly(siloxanes),poly(urethanes), poly(vinyl alcohols), poly(vinyl amines),poly(vinylmethylethers), poly(vinylpyrrolidones), silicones, celluloses,carbomethyl celluloses, hydroxypropyl methylcelluloses, chitins,chitosans, dextrans, dextrins, gelatins, hyaluronic acids andderivatives, functionalized hyaluronic acids, mannans, pectins,rhamnogalacturonans, starches, hydroxyalkyl starches, hydroxyethylstarches and other carbohydrate-based polymers, xylans, and copolymersthereof.

In another embodiment the at least one further biologically activemoiety in the form of a water-soluble controlled-release compound iscovalently conjugated through a stable linkage to an albumin-bindingmoiety. Preferably, said albumin-binding moiety is a C₈₋₂₄ alkyl moietyor fatty acid derivative. Preferred fatty acid derivatives are thosedisclosed in WO 2005/027978 A2 and WO 2014/060512 A1 which are herewithincorporated by reference.

Preferably, the at least one further biologically active moiety in theform of a water-soluble controlled-release comprises a biologicallyactive moiety selected from the group consisting of antihistamins; humananti-FGFR3 antibodies; soluble forms of human fibroblast growth factorreceptor 3; tyrosine kinase inhibitors; statins; CNP agonists; growthhormone; IGF-1; ANP; BNP; inhibitors of peptidases and proteases; andinhibitors of NPR-C.

A preferred antihistamin is meclozine.

A preferred tyrosine kinase inhibitor is NVP-BGJ398.

A preferred statin is rosuvastatin.

A preferred CNP agonist for the at least one further drug is vosoritide.

Preferred inhibitors of peptidases and proteases are NEP and furininhibitors.

A preferred inhibitor for NEP are thiorphan and candoxatril.

Preferred inhibitors of NPR-C are the fragment of SEQ ID NO:98(FGIPMDRIGRNPR) and antibody B701.

Preferred inhibitors of tyrosine kinases are as disclosed in U.S. Pat.Nos. 6,329,375 and 6,344,459, which are herewith incorporated byreference.

In one embodiment the at least one further biologically active moiety ordrug in the form of a water-soluble controlled-release comprises anantihistamin moiety or drug.

In another embodiment the at least one further biologically activemoiety or drug in the form of a water-soluble controlled-releasecomprises a human anti-FGFR3 antibody moiety or drug.

In another embodiment the at least one further biologically activemoiety or drug in the form of a water-soluble controlled-releasecomprises a soluble forms of human fibroblast growth factor receptor 3(sFGFR3) moiety or drug.

In another embodiment the at least one further biologically activemoiety or drug in the form of a water-soluble controlled-releasecomprises a tyrosine kinase inhibitor moiety or drug.

In another embodiment the at least one further biologically activemoiety or drug in the form of a water-soluble controlled-releasecomprises a statin moiety or drug.

In another embodiment the at least one further biologically activemoiety or drug in the form of a water-soluble controlled-releasecomprises a growth hormone moiety or drug. A preferred water-solublecontrolled-release growth hormone compound is compound 2 of example 2 ofWO2016/079114A1. Accordingly, a preferred water-solublecontrolled-release growth hormone compound has the following structure:

In another embodiment the at least one further biologically activemoiety or drug in the form of a water-soluble controlled-releasecomprises a CNP agonist moiety.

In another embodiment the at least one further biologically activemoiety or drug in the form of a water-soluble controlled-releasecomprises an IGF-1 moiety or drug.

In another embodiment the at least one further biologically activemoiety or drug in the form of a water-soluble controlled-releasecomprises an ANP moiety or drug.

In another embodiment the at least one further biologically activemoiety or drug in the form of a water-soluble controlled-releasecomprises a BNP moiety or drug.

In another embodiment the at least one further biologically activemoiety or drug in the form of a water-soluble controlled-releasecomprises an inhibitor of peptidases and proteases moiety or drug.

In another embodiment the at least one further biologically activemoiety or drug in the form of a water-soluble controlled-releasecomprises an inhibitor of NPR-C moiety or drug.

Another aspect of the present invention is the pharmaceuticalcomposition of the present invention for use as a medicament.

Another aspect of the present invention is the pharmaceuticalcomposition of the present invention for use in the treatment of apatient suffering from a disorder that benefits from stimulating growth.

Preferably, the patient is a mammalian patient, more preferably a humanpatient.

Preferably, such disorders that benefit from stimulating growth areselected from the group comprising achondroplasia, hypochondroplasia,short stature, dwarfism, osteochondrodysplasias, thanatophoricdysplasia, osteogenesis imperfecta, achondrogenesis, chondrodysplasiapunctata, homozygous achondroplasia, camptomelic dysplasia, congenitallethal hypophosphatasia, perinatal lethal type of osteogenesisimperfecta, short-rib polydactyly syndromes, rhizomelic type ofchondrodysplasia punctata, Jansen-type metaphyseal dysplasia,spondyloepiphyseal dysplasia congenita, atelosteogenesis, diastrophicdysplasia, congenital short femur, Langer-type mesomelic dysplasia,Nievergelt-type mesomelic dysplasia, Robinow syndrome, Reinhardtsyndrome, acrodysostosis, peripheral dysostosis, Kniest dysplasia,fibrochondrogenesis, Roberts syndrome, acromesomelic dysplasia,micromelia, Morquio syndrome, Kniest syndrome, metatrophic dysplasia,and spondyloepimetaphyseal dysplasia. Most preferably, the disorder thatbenefits from stimulating growth is achondroplasia.

Another aspect of the present invention is a method of treating apatient suffering from a disorder that benefits from stimulating growthby administering the pharmaceutical composition of the presentinvention.

Preferably, the patient is a mammalian patient, more preferably a humanpatient.

Preferably, such disorders that benefit from stimulating growth areselected from the group comprising achondroplasia, hypochondroplasia,short stature, dwarfism, osteochondrodysplasias, thanatophoricdysplasia, osteogenesis imperfecta, achondrogenesis, chondrodysplasiapunctata, homozygous achondroplasia, camptomelic dysplasia, congenitallethal hypophosphatasia, perinatal lethal type of osteogenesisimperfecta, short-rib polydactyly syndromes, rhizomelic type ofchondrodysplasia punctata, Jansen-type metaphyseal dysplasia,spondyloepiphyseal dysplasia congenita, atelosteogenesis, diastrophicdysplasia, congenital short femur, Langer-type mesomelic dysplasia,Nievergelt-type mesomelic dysplasia, Robinow syndrome, Reinhardtsyndrome, acrodysostosis, peripheral dysostosis, Kniest dysplasia,fibrochondrogenesis, Roberts syndrome, acromesomelic dysplasia,micromelia, Morquio syndrome, Kniest syndrome, metatrophic dysplasia,and spondyloepimetaphyseal dysplasia. Most preferably, the disorder thatbenefits from stimulating growth is achondroplasia.

If the CNP agonist is a polypeptide, such polypeptide may be prepared bystandard solid-phase peptide synthesis methods, e.g. by Boc chemistry(R. B. Merrifield, J. Am. Chem. Soc., 85(14): 2149-2154 (1963)).Alternatively, Fmoc (fluorenylmethoxycarbonyl) chemistry may beemployed.

Methods known in the art can be employed to improve purity and/or yield,including the use of pseudoproline or other dipeptide building blocks,fragment coupling and others (J. Wade et al., Lett. Pept. Sci.,7(2):107-112 (2000); Y. Fujiwara et al., Chem. Pharm. Bull.,44(7):1326-1331 (1996); P. Cherkupally et al., Eur. J. Org. Chem.,6372-6378 (2013)).

Alternatively, if the CNP agonist is a polypeptide, such polypeptide maybe produced by recombinant synthesis processes.

FIG. 1: Structure of CNP according to SEQ ID NO:1.

EXAMPLES

Materials and Methods

CNP SEQ ID No:1 was obtained from Bachem AG, Bubendorf, Switzerland(CNP-22, human, catalogue no. H-1296). CNP-34 SEQ ID No:40 and CNP-38SEQ ID No:24 were obtained from CASLO ApS, Kongens Lyngby, Denmark.

Side chain protected CNP-38 on CTC resin having Boc protected N-terminusand ivDde protected side chain of Lys26 (synthesized by Fmoc-strategy)was obtained from CASLO ApS, Kongens Lyngby, Denmark.

Side chain protected CNP-34 on TCP Tentagel resin having Boc protectedN-terminus and ivDde protected side chain of either Lys12, Lys16 orLys22 (synthesized by Fmoc-strategy) was obtained from Peptide SpecialtyLaboratories GmbH, Heidelberg, Germany. Side chain protected CNP-38 onTCP tentagel resin having free N-terminus (synthesized by Fmoc-strategy)was obtained from Peptide Specialty Laboratories GmbH, Heidelberg,Germany.

Methoxy PEG amine 5 kDa was obtained from Rapp Polymere GmbH, Tuebingen,Germany. All other PEGs used in this work were acquired from NOF EuropeN.V., Grobbendonk, Belgium.

Fmoc-N-Me-Asp(OtBu)—OH was obtained from Bachem AG, Bubendorf,Switzerland. S-Trityl-6-mercaptohexanoic acid was purchased fromPolypeptide, Strasbourg, France. HATU was obtained from MerckBiosciences GmbH, Schwalbach/Ts, Germany.

2,4-Dimethylbenzyl alcohol was obtained from abcr GmbH, Karlsruhe,Germany.

Fmoc-N-Me-Asp(OBn)-OH was obtained from Peptide International Inc.,Louisville, Ky., USA.

Neutral Endopeptidase (NEP) was obtained from Enzo Life Sciences GmbH,Lorrach, Germany.

All other chemicals and reagents were purchased from Sigma Aldrich GmbH,Taufkirchen, Germany.

Syringes equipped with polyethylene frits (MultiSynTech GmbH, Witten,Germany) were used as reaction vessels or for washing steps for peptideresins.

General Procedure for the Removal of ivDde Protecting Group from SideChain Protected CNPs on Resin

The resin was pre-swollen in DMF for 30 min and the solvent wasdiscarded. The ivDde group was removed by incubating the resin withDMF/hydrazine hydrate 4/1 (v/v, 2.5 mL/g resin) for 8×15 min. For eachstep fresh DMF/hydrazine hydrate solution was used. Finally, the resinwas washed with DMF (10 x), DCM (10 x) and dried in vacuo.

RP-HPLC Purification

For preparative RP-HPLC a Waters 600 controller and a 2487 DualAbsorbance Detector was used, equipped with the following columns:Waters XBridge™ BEH300 Prep C18 5 μm, 150×10 mm, flow rate 6 mL/min, orWaters XBridge™ BEH300 Prep C18 10 μm, 150×30 mm, flow rate 40 mL/min.Linear gradients of solvent system A (water containing 0.1% TFA v/v or0.01% conc. HCl v/v) and solvent system B (acetonitrile containing 0.1%TFA v/v or 0.01% conc. HCl v/v) were used.

HPLC fractions containing product were pooled and lyophilized if notstated otherwise.

Flash Chromatography

Flash chromatography purifications were performed on an Isolera Onesystem from Biotage AB, Sweden, using Biotage KP-Sil silica cartridgesand n-heptane and ethyl acetate as eluents. Products were detected at254 nm.

Analytical Methods

Analytical ultra-performance LC (UPLC)-MS was performed on a WatersAcquity system equipped with a Waters BEH300 C18 column (2.1×50 mm, 1.7μm particle size, flow: 0.25 mL/min; solvent A: water containing 0.04%TFA (v/v), solvent B: acetonitrile containing 0.05% TFA (v/v)) coupledto a LTQ Orbitrap Discovery mass spectrometer from Thermo Scientific orcoupled to a Waters Micromass ZQ.

Size exclusion chromatography (SEC) was performed using an AmershamBioscience AEKTAbasic system equipped with a Superdex 200 5/150 GLcolumn (Amersham Bioscience/GE Healthcare) equipped with a 0.45 μm inletfilter, if not stated otherwise. 20 mM sodium phosphate, 140 mM NaCl, pH7.4, was used as mobile phase.

Due to the reversible nature of the attachment of -L¹- to -Dmeasurements for NEP-stability and receptor affinity were made usingstable analogs of the CNP prodrugs of the present invention, i.e. theywere made using similar structures to those of the CNP prodrugs of thepresent invention which instead of a reversible attachment of —Z to -Dhave a stable attachment.

This was necessary, because the CNP prodrugs of the present inventionwould release CNP in the course of the experiment and said released CNPwould have influenced the result.

Quantification of Plasma Total CNP-38 Concentrations

Plasma total CNP-38 concentrations (conjugated and released CNP-38) weredetermined by quantification of the N-terminal signature peptide(sequence: LQEHPNAR) and C-terminal signature peptide (sequence:IGSMSGLGC) after tryptic digestion.

LC-MS analysis was carried out by using an Agilent 1290 UPLC coupled toan Agilent 6550 iFunnel Q-TOF mass spectrometer via an ESI probe.Chromatography was performed on a Waters Acquity BEH300 C18 analyticalcolumn (50×2.1 mm I.D., 1.7 μm particle size) with pre-filter at a flowrate of 0.25 mL/min (T=25° C.). Water (UPLC grade) containing 0.2%formic acid (v/v) was used as mobile phase A and acetonitrile (UPLCgrade) with 0.2% formic acid as mobile phase B. The gradient systemcomprised a short isocratic step at the initial parameters of 0.1% B for3.0 min followed by a linear increase from 0.1% B to 16% B in 17 min.Mass analysis was performed in the single ion monitoring (SIM) mode,monitoring the ions m/z 482.75 [M+2H]²⁺ (N-terminal) and m/z 824.36[M+H]¹⁺ (C-terminal). As internal standard deuterated CNP-38 peptide wasused.

Calibration standards of CNP-38 conjugate in blank plasma were preparedas follows: The thawed Li-heparin cynomolgous plasma was firsthomogenized, then centrifuged for 5 minutes. The CNP-38 conjugateformulation was diluted to a working solution of 10 gg/mL (conjugateCNP-38 eq.) in DMSO and spiked into blank plasma at concentrationsbetween 9.3 ng/100 μL (conjugate CNP-38 eq.) and 139.5 ng/100 μL(conjugate CNP-38 eq.). These solutions were used for the generation ofa calibration curve. Calibration curves were weighted 1/x² for bothsignature peptides (N- and C-Terminal). For quality control, threequality control samples were prepared accordingly with contents of 116.2ng/100 μL (high QC, conjugate CNP-38 eq.), 69.75 ng/100 μL (mid QC,conjugate CNP-38 eq.) and 23.25 ng/100 μL (low QC, conjugate CNP-38eq.).

For sample preparation, protein precipitation was carried out byaddition of 300 μL of pre-cooled (0° C.) methanol to 100 μL of theplasma sample. 200 μL of the supernatant were transferred into a newwell-plate and evaporated to dryness (under a gentle nitrogen stream at35° C.). 100 μL of reconstitution solvent (Thermo digestion buffer,order number 60109-101, Thermo Fisher Scientific GmbH, Dreieich,Germany) were used to dissolve the residue. 20 μg of trypsin (ordernumber V5111, Promega GmbH, Mannheim, Germany) were dissolved in 20 μLof 10 mM acetic acid. 2 μL of the trypsin solution were added to eachcavity.

After 4 hours incubation at 37° C. (water bath), 5 μL of a 0.5 M TCEPsolution were added to each cavity and incubated again for 5 min at 96°C. After the samples had cooled to room temperature, 3 μL acetonitrilewere added. The eluates were transferred into vials. 10 μL were injectedinto the UPLC-MS system.

Example 1

Synthesis of Linker Reagent 1f

Linker reagent 1f was synthesized according to the following scheme:

To a solution of N-methyl-N-Boc-ethylenediamine (2 g, 11.48 mmol) andNaCNBH₃ (819 mg, 12.63 mmol) in MeOH (20 mL) was added2,4,6-trimethoxybenzaldehyde (2.08 g, 10.61 mmol) portion wise. Themixture was stirred at rt for 90 min, acidified with 3 M HCl (4 mL) andstirred further 15 min. The reaction mixture was added to saturatedNaHCO₃ solution (200 mL) and extracted 5× with CH₂Cl₂. The combinedorganic phases were dried over Na₂SO₄ and the solvents were evaporatedunder reduced pressure. The resultingN-methyl-N-Boc-N′-Tmob-ethylenediamine 1a was dried in vacuo and used inthe next reaction step without further purification.

Yield: 3.76 g (11.48 mmol, 89% purity, 1a: double Tmob protectedproduct=8:1)

MS: m/z 355.22=[M+H]⁺, (calculated monoisotopic mass=354.21).

To a solution of 1a (2 g, 5.65 mmol) in CH₂Cl₂ (24 mL) COMU (4.84 g,11.3 mmol), N-Fmoc-N-Me-Asp(OBn)-OH (2.08 g, 4.52 mmol) and2,4,6-collidine (2.65 mL, 20.34 mmol) were added. The reaction mixturewas stirred for 3 h at rt, diluted with CH₂Cl₂ (250 mL) and washed 3×with 0.1 M H₂SO₄ (100 mEL) and 3× with brine (100 mL). The aqueousphases were re-extracted with CH₂Cl₂ (100 mL). The combined organicphases were dried over Na₂SO₄, filtrated and the residue concentrated toa volume of 24 mL. 1b was purified using flash chromatography.

Yield: 5.31 g (148%, 6.66 mmol)

MS: m/z 796.38=[M+H]⁺, (calculated monoisotopic mass=795.37).

To a solution of 1b (5.31 g, max. 4.52 mmol ref toN-Fmoc-N-Me-Asp(OBn)-OH) in THF (60 mL) DBU (1.8 mL, 3% v/v) was added.The solution was stirred for 12 min at rt, diluted with CH₂Cl₂ (400 mL)and washed 3× with 0.1 M H₂SO₄ (150 mEL) and 3× with brine (150 mL). Theaqueous phases were re-extracted with CH₂Cl₂ (100 mL). The combinedorganic phases were dried over Na₂SO₄ and filtrated. 1c was isolatedupon evaporation of the solvent and used in the next reaction withoutfurther purification.

MS: m/z 574.31=[M+H]⁺, (calculated monoisotopic mass=573.30).

1c (5.31 g, 4.52 mmol, crude) was dissolved in acetonitrile (26 mL) andCOMU (3.87 g, 9.04 mmol), 6-tritylmercaptohexanoic acid (2.12 g, 5.42mmol) and 2,4,6-collidine (2.35 mE, 18.08 mmol) were added. The reactionmixture was stirred for 4 h at rt, diluted with CH₂Cl₂ (400 mL) andwashed 3× with 0.1 M H₂SO₄ (100 mEL) and 3× with brine (100 mL). Theaqueous phases were re-extracted with CH₂Cl₂ (100 mL). The combinedorganic phases were dried over Na₂SO₄, filtrated and 1d was isolatedupon evaporation of the solvent. Product 1d was purified using flashchromatography.

Yield: 2.63 g (62%, 94% purity)

MS: m/z 856.41=[M+H]⁺, (calculated monoisotopic mass=855.41).

To a solution of 1d (2.63 g, 2.78 mmol) in i-PrOH (33 mL) and H₂O (11mL) was added LiOH (267 mg, 11.12 mmol) and the reaction mixture wasstirred for 70 min at rt. The mixture was diluted with CH₂Cl₂ (200 mL)and washed 3× with 0.1 M H₂SO₄ (50 mL) and 3× with brine (50 mL). Theaqueous phases were re-extracted with CH₂Cl₂ (100 mL). The combinedorganic phases were dried over Na₂SO₄, filtrated and 1e was isolatedupon evaporation of the solvent. 1e was purified using flashchromatography.

Yield: 2.1 g (88%)

MS: m/z 878.4=[M+Na]⁺, (calculated monoisotopic mass=855.40).

To a solution of 1e (170 mg, 0.198 mmol) in anhydrous DCM (4 mL) wereadded DCC (123 mg, 0.59 mmol), and a catalytic amount of DMAP. After 5min N-hydroxy-succinimide (114 mg, 0.99 mmol) was added and the reactionmixture was stirred at rt for 1 h. The reaction mixture was filtered,the solvent was removed in vacuo and the residue was taken up in 90%acetonitrile plus 0.1% TFA (3.4 mL). The crude mixture was purified byRP-HPLC. Product fractions were neutralized with 0.5 M pH 7.4 phosphatebuffer and concentrated. The remaining aqueous phase was extracted withDCM and 1f was isolated upon evaporation of the solvent.

Yield: 154 mg (81%)

MS: m/z 953.4=[M+H]⁺, (calculated monoisotopic mass=952.43).

Example 2

Synthesis of N^(εK4/εK10)-CNP Mono-Linker Thiol 2, N^(εK4)-CNPMono-Linker Thiol 2c and N^(εK10)-CNP Mono-Linker Thiol 2d

N^(εK4/εK10)-CNP mono-linker thiol (mixture of regioisomers with linkerconjugated at side chain amino group of Lys4 or Lys10) 2 is prepared bydissolving CNP-22 (5.2 μmol) in 0.6 mL DMSO. 0.15 mL 0.375 M boratebuffer, adjusted to pH 8.5 with tetrabutyl-ammoniumhydroxide hydrate, 60μL DIPEA and 1f (6.1 mg, 7.1 gmol) in 0.34 mL of DMSO are added and themixture is stirred for 30 min at rt. Reaction mixture is diluted with 2mL acetonitrile/water 1/1 (v/v) and 200 μL AcOH and the protectedN^(εK4/sεK10)-CNP mono-linker conjugate is isolated from the reactionmixture by RP-HPLC.

Optimized RP-HPLC gradients can be used for isolation of N^(εK4)-CNPmono-linker thiol 2a and N^(εK10)-CNP mono-linker thiol 2b.

Removal of protecting groups is affected by treatment of lyophilizedproduct fractions with 0.6 mL of 90/10/2/2 (v/v/v/v) HFIP/TFA/TES/waterfor 1h at rt. The deprotected N^(εK4/εK10) CNP mono-linker thiol 2 ispurified by RP-HPLC. Identity and purity of the product is determined byESI-LCMS.

Deprotected N^(εK4)-CNP mono-linker thiol 2c and N^(εK10)-CNPmono-linker thiol 2d can be obtained likewise from 2a and 2b,respectively.

Example 3

Synthesis of NG¹-CNP Mono-Linker Thiol 3

N^(αG1)-CNP mono-linker thiol 3 is prepared by dissolving CNP-22 (5.2gmol) in 0.6 mL DMSO. 0.25 mL 0.5 M phosphate buffer pH 7.4 and 1f (6.1mg, 7.1 μmol) in 0.34 mL of DMSO are added and the mixture is stirredfor several hours at rt. Reaction mixture is diluted with 2 mLacetonitrile/water 1/1 (v/v) and 200 μL AcOH and the protectedN^(αG1)-CNP mono-linker thiol is isolated from the reaction mixture byRP-HPLC.

Removal of protecting groups is affected by treatment of lyophilizedproduct fractions with 0.6 mL of 90/10/2/2 (v/v/v/v) HFIP/TFA/TES/waterfor 1h at rt. The deprotected N^(αG1)-CNP mono-linker thiol 3 ispurified by RP-HPLC. Identity and purity of the product is determined byESI-LCMS.

Example 4

PEGylation of CNP Mono-Linker Thiols 2c, 2d and 3

1 μmol CNP mono-linker thiol 2c is dissolved in 0.5 mL acetonitrile/0.2M succinate buffer pH 3.8 1/1 (v/v) 1.2 gmol of linear 40 kDaPEG-maleimide is added and the mixture is stirred at rt. The reaction isquenched by addition of 20 μL AcOH and CNP conjugate 4 is purified bypreparative RP-HPLC.

CNP conjugates 5 and 6 are prepared likewise from 1 μmol CNP mono-linkerthiols 2d and 3.

CNP content is determined by quantitative amino acid analysis aftertotal hydrolysis under acidic conditions.

Example 5

Release Kinetics In Vitro

CNP conjugates 4, 5 and 6 are dissolved in 60 mM sodium phosphate, 3 mMEDTA, 0.01% Tween-20, pH 7.4 at a concentration of approximately 2 mg/mLand filtered sterile. Mixtures are incubated at 37° C. At time pointsaliquots are withdrawn and analysed by RP-HPLC and ESI-MS. UV-signalscorrelating to liberated CNP are integrated and plotted againstincubation time.

Curve-fitting software is applied to estimate the corresponding halftimeof release.

Example 6

Pharmacokinetics and cGMP Production in Rats

Equimolar doses of CNP-22, CNP conjugates 4, 5 or 6 are injected iv andsc in normal rats.

Plasma CNP and cGMP levels over time are determined as described in theliterature (U.S. Pat. No. 8,377,884 B2).

Example 7

Synthesis of Dmb Protected 6-Mercaptohexanoic Acid 7

Compound 7 was synthesized according to the following scheme:

To a solution of 6-mercaptohexanoic acid (7.10 g, 47.90 mmol) intrifluoroacetic acid (20 mL), 2,4-dimethylbenzyl alcohol (13.5 g, 95.80mmol) was added. The mixture was stirred at rt for 60 min and then thetrifluoroacetic acid was removed in vacuo. The residue was dissolved ina mixture of 95.8 mL LiOH (3 M) and THF (81 mL) and stirred at rt for 60min. The solvent was removed in vacuo and the aqueous residue wasextracted 3x with EtOAc (200 mL). The combined organic phases were driedover MgSO₄, and the solvent was removed in vacuo. 7 was purified byRP-HPLC.

Yield: 2.27 g (8.52 mmol, 18%)

MS: m/z 267.01=[M+H]⁺, (calculated monoisotopic mass=266.13).

Example 8

Synthesis of Linker Reagent 8c

Linker reagent 8c was synthesized according to the following scheme:

To a solution of 1b (21.6 g, 27.18 mmol) in isopropanol (401 mL) wereadded water (130 mL) and LiOH (3.90 g, 163.06 mmol). The reactionmixture was stirred for 3 h at rt, then it was diluted with toluene (300mL) and washed 3× with 0.1 M HCl (200 mL). The combined aqueous phaseswere washed 3× with toluene (100 mL). The aqueous phase was basifiedwith 4 M NaOH (4 mL) to a pH of 8.5 and extracted 8× with CH₂Cl₂ (200mL). The combined CH₂Cl₂ phases were washed with brine (50 mL), driedover Na₂SO₄. 8b was isolated upon evaporation of the solvent and used inthe next reaction without further purification.

Yield: 11.89 g (24.59 mmol, 90%)

MS: m/z 484.16=[M+H]⁺, (calculated monoisotopic mass=483.26).

To a solution of 7 (293 mg, 1.10 mmol) and PyBOP (572 mg, 1.10 mmol) inTHF (10 mL) was added DIEA (0.52 mL, 3.00 mmol) under a N₂-atmosphere.The reaction mixture was stirred for 60 min at rt. A solution of 8b (484mg, 1.00 mmol) in THF (2 mL) was added and the reaction was stirred fora further 60 min. The reaction was quenched with 2 M citric acidsolution (10 mL) and the THF was removed in vacuo. The resulting aqueousphase was then extracted 2× with EtOAc (15 mL) and the combined organiclayers were washed with water (10 mL) and brine (10 mL), and dried overMgSO₄. The solvent was removed in vacuo and 8c was purified by RP HPLC.

Yield: 330 mg (0.451 mmol, 45%)

MS: m/z 732.34=[M+H]⁺, (calculated monoisotopic mass=731.38).

Example 9

Synthesis of Linker Reagent 9

Linker reagent 9 was synthesized according to the following scheme:

To a solution of 8b (2.00 g, 4.14 mmol) and Fmoc-Cl (1.07 g, 4.14 mmol)in dioxane (20 mL) was added 1 M Na₂CO₃ solution (20 mL). The reactionmixture was stirred for 40 min at rt. Water (100 mL) and diethyl ether(100 mL) were added and the aqueous phase was extracted 2× with diethylether (100 mL). The aqueous phase was acidified with conc. HCl until pH1 and again extracted 3× with diethyl ether. The combined organic phaseswere dried over Na₂SO₄ and the solvent was removed in vacuo. 9 was usedin the next step without further purification.

Yield: 2.63 g (3.73 mmol, 90%)

MS: m/z 728.32=[M+Na]⁺, (calculated monoisotopic mass=705.33).

Example 10

Synthesis of Reversible Lys26 CNP-38 PEG2×20 kDa Conjugate 10f

Conjugate 10f was synthesized according to the following scheme:

2.00 g (0.21 mmol) of side chain protected CNP-38 on CTC resin havingBoc protected N-terminus and ivDde protected side chain of Lys26 wasivDde deprotected according to the procedure given in Materials andMethods to obtain 10a. A solution of linker reagent 8c (336 mg, 0.46mmol), PyBOP (239 mg, 0.46 mmol) and DIEA (182 μL, 1.04 mmol) in DMF (5mL) was incubated for 10 min at rt, then added to the resin 10a. Thesuspension was shaken for 2 h at rt. The resin was washed 10× with DMF(10 mL) and 10× with DCM (10 mL) and dried in vacuo for 15 min. Cleavageof the peptide from resin and removal of protecting groups was achievedby treatment of the resin with 15 mL pre-cooled (−18° C.) cleavagecocktail 68.5/10/10/5/3.5/1 (v/w/v/v/v/v)TFA/DTT/thioanisole/phenol/water/TIPS. The mixture was allowed to warmto rt and was agitated for 60 min. The resin was filtered off and crude10c was precipitated in pre-cooled diethyl ether (−18° C.). Theprecipitate was dissolved in ACN/water and purified by RP-HPLC. Thecombined HPLC fractions were used directly in the next step.

MS: m/z 1124.60=[M+4H]⁴⁺, (calculated monoisotopic mass for[M+4H]⁴⁺=1124.59).

To the combined HPLC fractions of 10c (250 mL) 40 mL of 0.5 M citricacid buffer (pH=5.00) and 7 mL of a 0.01 M solution of2,2′-dithiobis(pyridine-N-oxide) solution in 1/1 (v/v)acetonitrile/water were added. After incubation for 5 min at rt thereaction was complete. The mixture was diluted with 500 mL watercontaining 0.1% TFA (v/v) and acidified with AcOH (20 mL) to a pH ofapprox. 2. 10d was purified by RP-HPLC.

Yield: 101 mg (17.3 gmol, 9%) CNP-38-linker-Dmb*10 TFA

MS: m/z 1124.10=[M+4H]⁴⁺, (calculated monoisotopic mass for[M+4H]⁴⁺=1124.09).

Cleavage of the Dmb protecting group was achieved by adding 30 mLpre-cooled (−18° C.) cleavage cocktail 100/5/3/2/1 (v/v/w/v/v)TFA/MSA/DTT/water/thioanisole to 10d (101 mg, 17.3 μmol) and stirringfor 3 h at 0° C. Crude 10e was precipitated in pre-cooled (−18° C.)diethyl ether. The precipitate was dissolved in water containing 0.1%TFA (v/v) and incubated for 10 min in order to hydrolyze any TFA esters.10e was purified by RP-HPLC.

Product fractions were combined and freeze dried.

Yield: 46 mg (8.34 gmol, 48%) CNP-38-linker-thiol*10 TFA

MS: m/z 1094.58=[M+4H]⁴⁺, (calculated monoisotopic mass for[M+4H]⁴+=1094.57).

To a solution of 10e (46 mg, 8.43 μmol) in 1.15 mL water containing 0.1%TFA (v/v) was added a solution of PEG 2×20 kDa maleimide (SunbrightGL2-400MA, 870 mg, 21.75 gmol) in 4.35 mL water containing 0.1% TFA(v/v), followed by 0.5 M lactic acid buffer (1.07 mL, pH=4.20). Themixture was stirred at rt for 4 h. Conjugate 10f was purified byRP-HPLC.

Yield: 233 mg (5.21 μmol, 62%) conjugate 10f*10 HCl

Example 11

Synthesis of Reversible Lys26 CNP-38 PEG4×10 kDa Conjugate 11i

Conjugate 11i was synthesized according to the following scheme:

To a solution of 9 (353 mg, 0.50 mmol) and PyBOP (260 mg, 0.50 mmol) inDMF (9 mL) was added DIEA (105 μL, 0.60 mmol).This mixture was drawnonto Lys26-side-chain deprotected CNP-38 resin 10a (2.00 g, 0.21 mmol)and the suspension was shaken for 2 h at rt in order to afford resin11a. The resin was washed 10× with DMF (7 mL). Cleavage of the Fmocprotecting group in 11a was carried out with a solution of HOBt (0.68 g,5.03 mmol) and piperazine (3.00 g, 34.83 mmol) in DMF (47 mL).Therefore, the resin was incubated 5× with 10 mL of the cleavage mixturefor 15 min at rt each time. Then, the resin was washed 7× with DMF (7mL).

A solution of Fmoc-Lys(Fmoc)-OH (449 mg, 0.76 mmol), COMU (325 mg, 0.76mmol) and DIEA (165 μL, 0.95 mmol) in DMF (9 mL) was prepared and drawnonto the resin. The mixture was shaken for 2 h at rt. The procedure wasrepeated twice, each for 1 h with freshly prepared coupling mixture. Theresin was washed 10× with DMF (7 mL) and the remaining free amino groupswere capped with 8 mL 1/1/2 (v/v/v) Ac₂O/pyridine/DMF.

Cleavage of the Fmoc protecting groups in 11c was carried out with asolution of HOBt (0.68 g, 5.03 mmol), piperazine (3.00 g, 34.83 mmol) inDMF (47 mL). Therefore, the resin was incubated 5× with 10 mL of thecleavage mixture for 15 min at rt each time. The resin was washed 7×with DMF (7 mL).

To a solution of 7 (266 mg, 1.00 mmol) and PyBOP (520 mg, 1.00 mmol) inDMF (9 mL) was added DIEA (209 μL, 1.20 mmol). This mixture was drawnonto the resin and was shaken for 2 h at rt. The resin was washed 7×with DMF (7 mL) affording resin lie. Cleavage of the peptide from resinand removal of protecting groups was achieved by treatment of the resinwith 15 mL pre-cooled (−18° C.) cleavage cocktail 68.5/10/10/5/3.5/1(v/w/v/v/v/v) TFA/DTT/thioanisole/phenol/water/TIPS. The mixture wasallowed to warm to rt and was agitated for 3 h at rt. The resin wasfiltered off and crude 11f was precipitated in pre-cooled (−18° C.)diethyl ether and purified by RP-HPLC. The combined HPLC fractions wereused directly in the next step.

MS: m/z 1218.66=[M+4H]⁴⁺, (calculated monoisotopic mass for[M+4H]⁴+1218.65).

To the combined HPLC product fractions of 11f (1 L) 160 mL of 0.5 Mcitric acid buffer (pH=5.00) and 100 mL of a 50 mM2,2′-dithiobis(pyridine-N-oxide) solution in 9/1 (v/v)acetonitrile/water were added. The mixture was stirred for 4 h at rt andthen diluted with 1 L of water containing 0.1% TFA (v/v). 11g waspurified by RP-HPLC. The product fractions were combined andlyophilized.

Yield: 64.3 mg (10.7 μmol, 6%) CNP-38-linker-DMB*10 TFA

MS: m/z 1218.15=[M+4H]⁴⁺, (calculated monoisotopic mass for[M+4H]⁴⁺=1218.14).

Cleavage of the Dmb protecting group was achieved by adding 45 mL ofpre-cooled (−18° C.) cleavage cocktail 100/5/3/2/1 (v/v/w/v/v)TFA/MSA/DTT/water/thioanisole to 11g (61.8 mg, 10.3 μmol), and thenstirring for 4 h at 0° C. Crude 11h was precipitated in pre-cooled (−18°C.) ether. The precipitate was dissolved in a solution of 1/1 (v/v)acetonitrile/water containing 0.1% TFA (v/v) and incubated for 4 h at rtin order to hydrolyze any TFA esters. 11h was purified by RP-HPLC.

Yield: 38.4 mg (6.65 μmol, 65%) CNP-38-linker-thiol*10 TFA

MS: m/z 1159.11=[M+4H]⁴⁺, (calculated monoisotopic mass for[M+4H]⁴⁺=1159.10).

To a solution of 11h (34.6 mg, 5.99 μmol) in 1 mL water containing 0.1%TFA (v/v) was added a solution of PEG 2×10 kDa maleimide (SunbrightGL2-200MA, 1.12 g, 56.03 μmol) in 6.1 mL water containing 0.1% TFA(v/v), followed by 0.5 M lactic acid buffer (1.46 mL, pH=4.00). Themixture was stirred at rt for 4 h. Conjugate 11i was purified byRP-HPLC.

Yield: 227 mg (4.96 μmol, 83%) conjugate 11i*10 HCl

Example 12

Synthesis of Permanent Lys26 CNP-38 PEG4×10 kDa Conjugate 12g

Conjugate 12g was synthesized according to the following scheme:

To a solution of Fmoc-Lys(Fmoc)-OH (365 mg, 0.62 mmol) and PyBOP (322mg, 0.62 mmol) in DMF (4.6 mL) was added DIEA (0.11 mL, 0.62 mmol).Themixture was drawn onto resin 10a (2.0 g, 0.21 mmol). The suspension wasshaken for 2 h at rt. The resin was washed 10× with DMF (7 mL). Cleavageof the Fmoc protecting groups in 12a was carried out with a solution ofHOBt (1.35 g, 9.99 mmol), piperazine (6.00 g, 69.66 mmol) in DMF (94mL). Therefore, the resin was incubated 5× with the cleavage mixture for15 min at rt each time, affording resin 12b. Then the resin was washed7× with DMF (7 mL).

To a solution of 7 (283 mg, 1.06 mmol) and PyBOP (552 mg, 1.06 mmol) inDMF (6.5 mL), DIEA (185 μL, 1.06 mmol) was added and the mixture wasdrawn onto resin 12b (2.07 g, 0.10 mmol/g, 0.21 mmol). The mixture wasshaken for 2 h at rt. Then, the resin was washed 10× each with DMF (7mL) and CH₂Cl₂ (7 mL) and dried in vacuo.

Cleavage of the peptide from resin and removal of protecting groups wasachieved by treatment of the resin with 15 mL pre-cooled (−18° C.)cleavage cocktail 68.5/10/10/5/3.5/1 (v/w/v/v/v/v)TFA/DTT/thioanisole/phenol/water/TIPS. The mixture was allowed to warmto rt and was agitated for 2.5 h. The resin was filtered off and crude12d was precipitated in pre-cooled diethyl ether (−18° C.) and purifiedby RP-HPLC. The combined HPLC fractions were used directly in the nextstep.

MS: m/z 1172.37=[M+4H]⁴⁺, (calculated monoisotopic mass for[M+4H]⁴+=1172.37).

To the combined HPLC product fractions of 12d (390 mL) 58.5 mL of 0.5 Mcitric acid buffer (pH=5.00) and 8.9 mL of a 10 mM2,2′-dithiobis(pyridine-N-oxide) solution in 1/1 (v/v)acetonitrile/water were added. The mixture was stirred for 10 min at rtthen diluted with 400 mL of water containing 0.1% TFA (v/v). 12e waspurified by RP-HPLC.

Yield: 100 mg (17.5 μmol, 8% over 6 steps) CNP-38-linker-Dmb*9 TFA

MS: m/z 1171.87=[M+4H]⁴⁺, (calculated monoisotopic mass for[M+4H]⁴+=1171.86).

Cleavage of the Dmb protecting group was achieved by adding 65 mLpre-cooled (−18° C.) cleavage cocktail 100/5/3/2/1 (v/v/w/v/v)TFA/MSA/DTT/water/thioanisole to 12e (100 mg, 17.5 μmol) and stirringfor 3.5 h at 0° C. Crude 12f was precipitated in pre-cooled (−18° C.)diethyl ether. The precipitate was dissolved in water containing 0.1%TFA (v/v) and incubated for 2 h at rt in order to hydrolyze any TFAesters. 12f was purified by RP-HPLC.

Yield: 43.4 mg (7.92 μmol, 45%) CNP-38-linker-thiol*9TFA

MS: m/z 1112.83=[M+4H]⁴⁺, (calculated monoisotopic mass for[M+4H]⁴⁺=1112.82).

To a solution of 12f (39.6 mg, 7.22 μmol) in 1 mL water containing 0.1%TFA (v/v) was added a solution of PEG 2×10 kDa maleimide (SunbrightGL2-200MA, 1.22 g, 59.94 μmol) in 6.16 mL water containing 0.1% TFA(v/v), followed by 0.5 M lactic acid buffer (1.41 mL, pH=4.20). Themixture was stirred at rt for 4 h. Conjugate 12g was purified byRP-HPLC.

Yield: 204 mg (4.48 μmol, 57%) conjugate 12g*9 HCl

Example 13

Synthesis of PEG5 kDa Thiol 13c

PEG5 kDa thiol 13c was synthesized according to the following scheme:

To a solution of 13b (58.6 mg, 0.15 mmol), HOBt (22.9 mg, 0.15 mmol) andEDC hydrochloride (28.8 mg, 0.15 mmol) in DCM (1.00 mL) 2,4,6-collidine(121 mg, 1.00 mmol) was added. Then, a solution of methoxy PEG amine 5kDa 13a (500 mg, 0.10 mmol) in DCM (4.00 mL) was added and the mixturewas stirred for 16 h at rt. The solvent was evaporated and the mixturewas dissolved in ACN/water and purified by RP-HPLC. The amount ofsolvent was reduced in vacuo and the aqueous residue was extracted withDCM (1×100 mL, 2×50 mL). The combined organic layers were reduced invacuo to 20 mL. TFA (1.6 mL) and TES (3.5 mL) were added and the mixturewas stirred at rt for 4.5 h. 13c was precipitated in diethyl ether,stored over night at −20° C., filtered and dried in vacuo.

Yield: 372 mg (72 μmol, 72%)

Example 14

Synthesis of Permanent N-Terminal CNP-34 PEG 5 kDa Conjugate 14e

Conjugate 14e was synthesized according to the following scheme:

Side chain protected CNP-34 on TCP tentagel resin having free N-terminus14a (0.78 g, 70 μmol) was pre-swollen in DMF for 30 min. A solution ofmaleimido hexanoic acid (85.3 mg, 0.40 mmol), DIC (50.9 mg, 0.40 mmol)and Oxyma (57.4 mL, 0.40 mmol) in DMF (6 mL) was drawn onto the resinand the mixture was shaken for 30 min at rt. The coupling then wasrepeated once with freshly prepared coupling solution. The resin waswashed 10× each with DMF and CH₂Cl₂ and dried in vacuo affording 14b.

Cleavage of the peptide from resin and removal of protecting groups wasachieved by treatment of the resin with 6 mL cleavage cocktail 100/3/2/1(v/v/v/v) TFA/TES/water/thioanisole for 1.5 h at rt. The crude peptidewas precipitated in pre-cooled (−18° C.) diethyl ether.

MS: m/z 937.77=[M+4H]⁴⁺, (calculated monoisotopic mass for[M+4H]⁴+=937.74).

The precipitate was dissolved in 15 mL TFA. A solution ofdiphenylsulfoxide (68.06 mg, 0.34 mmol) and anisole (0.18 mL, 1.68 mmol)in 5 mL TFA was added. Trichloromethylsilane (0.47 mL, 4.17 mmol) wasadded and the mixture was stirred for 15 min at rt. Ammonium fluoride(0.38 g, 10.3 mmol) was added and the solution was agitated for afurther 2 min. The crude material was precipitated in pre-cooled (−18°C.) diethyl ether and purified by RP-HPLC affording 14d.

Yield: 8.30 mg (1.78 μmol, 82% purity, 1.4% over 3 steps) CNP-34-Malhx*8TFA

MS: m/z 937.26=[M+4H]⁴⁺, (calculated monoisotopic mass for[M+4H]⁴+=937.23).

To a solution of 14d (7.34 mg, 1.57 μmol) in 200 μL 1/1 (v/v)acetonitrile/water containing 0.1% TFA (v/v) was added a solution of 13c(20 mg, 3.90 μmol) in 200 μL water containing 0.1% TFA (v/v), followedby 200 μL 0.5 M acetate buffer (pH=5.00). The mixture was incubated atrt for 30 min. Conjugate 14e was purified by RP-HPLC.

Yield: 9.92 mg (1.01 μmol, 57%) conjugate 14e*8 TFA

Example 15

Synthesis of Permanent N-Terminal CNP-38 PEG 5 kDa Conjugate 15e

Conjugate 15e was synthesized according to the following scheme:

Compound 15d was synthesized as described for 14d, except that sidechain protected CNP-38 on TCP tentagel resin having free N-terminus 15a(1.34 g, 0.12 mmol) was used as starting material.

Yield: 15.6 mg (2.94 μmol, 6.6%) CNP-38-Malhx*9 TFA

MS: m/z 1064.05=[M+4H]⁴⁺, (calculated monoisotopic mass for[M+4H]⁴+=1064.04).

Conjugate 15e was synthesized as described for 14e, except that 15d(8.34 g, 1.58 mmol) was used as starting material.

Yield: 9.47 mg (0.91 μmol, 31%) conjugate 15e*9 TFA

Example 16

Synthesis of Permanent Lys12 CNP-34 PEG 5 kDa Conjugate 16e

Conjugate 16e was synthesized according to the following scheme:

1.00 g (0.10 mmol) of side chain protected CNP-34 on TCP tentagel resinhaving Boc protected N-terminus and ivDde protected side chain of Lys12was ivDde deprotected according to the procedure given in Materials andMethods to obtain 16a.

Compound 16d was synthesized as described for 14d, except that resin 16a(1.00 g, 0.10 mmol) was used as starting material.

Yield: 17.0 mg (3.65 μmol, 3.7%) CNP-34-Lys12-Malhx*8 TFA

MS: m/z 937.25=[M+4H]⁴⁺, (calculated monoisotopic mass for[M+4H]⁴+=937.23).

Conjugate 16e was synthesized as described for 14e, except that 16d (17mg, 3.65 μmol) was used as starting material.

Yield: 12.2 mg (1.25 μmol, 34%) conjugate 16e*8 TFA

Example 17

Synthesis of Permanent Lys16 CNP-34 PEG 5 kDa Conjugate 17e

Conjugate 17e was synthesized according to the following scheme:

0.78 g (0.07 mmol) of side chain protected CNP-34 on TCP tentagel resinhaving Boc protected N-terminus and ivDde protected side chain of Lys16was ivDde deprotected according to the procedure given in Materials andMethods to obtain 17a.

Compound 17d was synthesized as described for 14d, except that resin 17a(0.78 g, 0.13 mmol) was used as starting material.

Yield: 5.39 mg (1.16 μmol, 1.7%) CNP-34-Lys16-Malhx*8 TFA

MS: m/z 937.26=[M+4H]⁴⁺, (calculated monoisotopic mass for[M+4H]⁴+=937.23).

Conjugate 17e was synthesized as described for 14e, except that 17d(5.39 mg, 1.16 μmol) was used as starting material.

Yield: 10.7 mg (1.09 μmol, 94%) conjugate 17e*8 TFA

Example 18

Synthesis of Permanent Lys22 CNP-34 PEG 5 kDa conjugate 18e

Conjugate 18e was synthesized according to the following scheme:

1.07 g (0.11 mmol) of side chain protected CNP-34 on TCP tentagel resinhaving Boc protected N-terminus and ivDde protected side chain of Lys22was ivDde deprotected according to the procedure given in Materials andMethods to obtain 18a.

Compound 18d was synthesized as described for 14d, except that resin 18a(1.07 g, 0.11 mmol) was used as starting material.

Yield: 5.20 mg (1.12 μmol, 1.0%) CNP-34-Lys22-Malhx*8 TFA

MS: m/z 937.26=[M+4H]⁴⁺, (calculated monoisotopic mass for[M+4H]⁴+=937.23).

Conjugate 18e was synthesized as described for 14e, except that 18d (5.2mg, 1.12 μmol) was used as starting material.

Yield: 4.20 mg (0.43 μmol, 38%) conjugate 18e*8 TFA

Example 19

Synthesis of Permanent Lys26 CNP-38 PEG 5 kDa Conjugate 19e

Conjugate 19e was synthesized according to the following scheme:

(0.865 g, 0.10 mmol) of side chain protected CNP-38 on TCP tentagelresin having Boc protected N-terminus and ivDde protected side chain ofLys26 was ivDde deprotected according to the procedure given inMaterials and Methods to obtain 19a.

Compound 19d was synthesized as described for 14d, except that resin 19a(0.865 g, 0.10 mmol) was used as starting material.

Yield: 10.3 mg (1.95 μmol, 2.0%) CNP-38-Lys26-Malhx*9 TFA

MS: m/z 1064.05=[M+4H]⁴⁺, (calculated monoisotopic mass for[M+4H]⁴⁺=1064.04).

Conjugate 19e was synthesized as described for 14e, except that 19d(4.70 mg, 1.10 μmol) was used as starting material.

Yield: 3.20 mg (0.31 μmol, 28%) conjugate 19e*9 TFA

Example 20

Release Kinetics In Vitro

CNP conjugates 10f and 11i were dissolved in a PBS buffer containing 3mM EDTA and 10 mM methionine, pH 7.4 at a concentration of approximately1 mg conjugate/mL. The solutions was filtered sterile and were incubatedat 37° C. At time points aliquots were withdrawn and analysed by RP-HPLCand ESI-MS. UV-signals correlating to liberated CNP were integrated andplotted against incubation time.

Curve-fitting software was applied to estimate the correspondinghalftime of release.

Results:

For conjugate 10f a release half life time of 8.5 d (+1 d) was obtained.

For conjugate 11i a release half life time of 9.5 d (+1.5 d) wasobtained.

Example 21

Functional cGMP Stimulation in NIH-3T3 Cells with CNP Variants

Functional activity of CNP variants were determined in a cell-basedassay with NIH-3T3 cells (Murine Embryo Fibroblast cell line). Thesecells express endogenously the NPR-B receptor on the cell surface.Stimulation of the NPR-B receptor with CNP leads to intracellularproduction of the second messenger cGMP which is detected with acommercially available cGMP assay. NIH-3T3 cells were routinely culturedin DMEM F-12 medium with 5% FBS and 5 mM glutamine at 37° C. and 5% CO₂.For each assay, 50,000 cells were resuspended in stimulation buffer(Dulbecco's PBS with IBMX) and incubated with the CNP variants indifferent concentrations. CNP (dilutions were made in PBS with 0.2%BSA). After incubation of 30 min at 37° C. and 5% CO₂, the cells werelyzed and cGMP levels were determined with a commercially available cGMPTR-FRET assay (Cisbio, cGMP kit, Cat. No. 62GM2PEB). PEGylated CNPvariants were always characterized in comparison with the non-PEGylatedversion in the same experiment batch. If possible, evaluation of theresidual activity was done via the relative EC₅₀- parameter of theresulting dose-response curve (restricted model with common slope).

TABLE 1 Residual NPR-B activity of PEGylated CNP variants in a cell-based assay as determined against the non-PEGylated CNP variant CNP EC₅₀compound/ Compound Variant PEGylation EC₅₀ CNP-38 15e CNP-38 5 kDa PEG,N-Terminus >5 19e CNP-38 5 kDa PEG, Lys26 >100 12g CNP-38 4 × 10 kDaPEG, Lys26 >>100 11i CNP-38 4 × 10 kDa PEG, Lys26 >>100 10f CNP-38 2 ×20 kDa PEG, Lys26 >>100

Comparing the tested PEG attachment sites, the attachment at the Lys26(ring-lysine) showed the highest functional activity reduction, whereasthe N-terminal attachment showed relatively high residual functionalactivity values. Increasing the PEG size resulted in a better shieldingof the CNP molecule and a lower residual functional activity.

Example 22

Growth Study in FVB Mice after 5 Weeks Treatment with CNP-38 by DailySubcutaneous Bolus Injection or by Continuous Subcutaneous Infusion

This study was performed in order to test the effect of dailysubcutaneous bolus injection vs. continuous subcutaneous infusion ofCNP-38 on animal growth. 21- to 22-days-old wild-type FVB male mice(n=9/group) were given 50 nmol/kg/d CNP-38 or vehicle (30 mM acetate pH4 containing 5% sucrose and 1% benzylic alcohol) either by dailysubcutaneous bolus injection or by continuous subcutaneous infusion inthe scapular region over 35 days. Continuous infusion was applied byAlzet osmotic pumps model 1002 for week 1-2, followed by model 1004 forweek 3-5. CNP-38 concentrations in the pumps were adjusted for the meananimal weight at study day 7 (pump model 1002) or study day 25 (pumpmodel 1004). Growth was determined at d 35 by total body lengthmeasurement and X-ray measurements of the right femur and tibia.

Results of animals treated by daily subcutaneous bolus injection: At d35, total body length of CNP-38 treated animals was 110.2%, right femurlength was 105.6% and right tibia length was 104.0% compared to vehicletreated animals.

Results of animals treated by continuous subcutaneous infusion: At d 35,total body length of CNP-38 treated animals was 121.7%, right femurlength was 107.5% and right tibia length was 112.2% compared to vehicletreated animals.

It was concluded that continuous subcutaneous infusion or related slowrelease formulations of CNP-38 (e.g. a slow releasing CNP-38 prodrug)are at least as effective as daily subcutaneous bolus injection ineliciting growth in the appendicular and axial skeleton.

Example 23

Pharmacokinetic Study of Permanent Lys26 CNP-38 PEG4×10 kDa Conjugate12g in Cynomolgus Monkeys

This study was performed in order to show the suitability of 12g as amodel compound for a slow release CNP-38 prodrug in cynomolgus monkeys.Male cynomolgus monkeys (2-4 years old, 3.5-4.1 kg) received either asingle intravenous (n=3 animals) or a single subcutaneous (n=2 animals)administration of 12g at a dose of 0.146 mg CNP-38 eq/kg. Blood sampleswere collected up to 168 h post dose, and plasma was generated. Plasmatotal CNP-38 concentrations were determined by quantification of theN-terminal signature peptide (sequence: LQEHPNAR) and C-terminalsignature peptide (sequence: IGSMSGLGC) after tryptic digestion asdescribed in Materials and Methods.

Results: Dose administrations were well tolerated with no visible signsof discomfort during administration and following administration. Nodose site reactions were observed any time throughout the study. Afterintraveneous injection the CNP-38 t_(max) was observed at 15 min(earliest time point analyzed), followed by a slow decay in CNP-38content with a half life time of approx. 24 h. After subcutaneousinjection the CNP-38 concentration peaked at a t_(max) of 48 h. At 168 hthe CNP-38 concentration was still as high as ca. 50% of c_(max). Thebioavailability was ca. 50%.

Similar PK curves were obtained for the N- and the C-terminal signaturepeptide up to 168 h post dose, indicating the presence of intact CNP-38in the conjugate.

The favourable long lasting PK over several days and the stability ofCNP-38 in the conjugate indicates the suitability of the permanent modelcompound Lys26 CNP-38 PEG 4×10 kDa conjugate 12g as a slow releasingCNP-38 prodrug after subcutaneous injection. It can be concluded thatsimilar conjugates having a transiently Lys26 linked CNP-38 (like e.g.11i) are suitable CNP-38 prodrugs providing long lasting levels ofreleased bioactive CNP-38 over several days.

Example 24

Digest of CNP Variants by Neutral Endopeptidase In Vitro

In order to determine the in vitro stability of various CNP variantsincluding different peptide chain lengths and PEGylations usingdifferent PEGylation sites and PEG molecules in the presence of NeutralEndopeptidase (NEP), a NEP digest assay was established. This assaymonitored the decrease of the non-digested CNP variant (normalized withthe internal standard PFP) over time in reference to the t₀-time point.

In detail, recombinant human NEP (2.5 μg/mL final concentration) and thestandard pentafluorophenol (PFP; 40 μg/mL final concentration) wereadded to the CNP variant (100 μg CNP equivalents/mL) in digest buffer(50 mM Tris-HCl, pH 7.4, 10 mM NaCl). The solution was incubated at 37°C. and 500 rpm for up to 4 days. Samples were taken at different timeintervals. The reaction was stopped by a combined reduction and heatdenaturation adding TCEP (tris(2-carboxyethyl)phosphine; 25 mM finalconcentration) and incubating the mixture at 95° C., 500 rpm for 5minutes. The resulting reaction products were assigned using HPLC-MS.The half life of each CNP variant was calculated via the ratio change inthe HPLC-UV peak areas of CNP and PFP over time. To compensate forvariations in the protease activity, a CNP-38 or CNP-34 digest wascarried out in every batch measurement as reference.

Table 2 lists the half-lives, based on the in vitro NEP cleavage assay,of various CNP variants of different lengths and having various PEGmolecules attached to different side chains.

CNP- half life Compound variant PEGylation norm. [h] CNP-22¹ CNP-22 — 0.32 CNP-34¹ CNP-34 —  4.15 14e¹ CNP-34 5 kDa PEG, N-Terminus Almost noproteolysis after 4 days. 17e¹ CNP 34 5 kDa PEG, Lys16 54.23 18e¹ CNP-345 kDa PEG, Lys22 38.87 16e¹ CNP-34 5 kDa PEG, Lys12 No evaluationpossible. CNP-38² CNP-38 — 12.10 19e² CNP-38 5 kDa PEG, Lys26 62.76 15e²CNP-38 5 kDa PEG, N-Terminus Almost no proteolysis after 4 days. 12g²CNP-38 4 × 10 kDa PEG, -Lys26 Almost no proteolysis after 4 days. ¹Dueto variations in NEP catalytic activity between experiments, a mean wasformed of all CNP-34 half life measurements (4.15 h) and the CNP-34conjugates' half life measurements were normalized to this mean using acoefficient to calculate the adjusted t_(1/2). ²Due to variations in NEPcatalytic activity between experiments, a mean was formed of all CNP-38half life measurements (12.10 h) and the CNP-38 conjugates' half lifemeasurements were normalized to this mean using a coefficient tocalculate the adjusted t_(1/2).

The rank order of resistance towards NEP is as follows: The longerCNP-variant (CNP-38) is more stable than the shorter CNP variant(CNP-34), which in turn is more stable than the shorter CNP-22. Theorder of the PEG-attachment sites is as follows:N-terminal>next-to-ring>ring. Therefore, an N-terminal PEG attachmentconfers the highest stability towards the proteolytic digest with NEPfor the tested conjugates. The stability of CNP-38 PEGylated at Lys26can be increased with increasing PEG size.

Example 25

Pharmacokinetic Study of Transient Conjugates 10f and 11i in CynomolgusMonkeys

This study was performed in order to show the suitability of 10f and 11ias slow release CNP-38 prodrugs in cynomolgus monkeys. Male cynomolgusmonkeys (2-4 years old, 3-5 kg) received either a single subcutaneousadministration (n=3 animals) of compound 10f or a single subcutaneous(n=3 animals) administration of 11i at a dose of 0.146 mg CNP-38 eq/kg.Blood samples were collected up to 168 h post dose, and plasma wasgenerated. Plasma levels of total CNP-38 content were analyzed asdescribed in example 24. In order to analyze the plasma content of freeCNP-38, the blood samples were acidified after withdrawal by adding 20vol % of 0.5 M sodium citrate buffer pH 4 to stop further CNP-38 releasefrom the conjugate. Free CNP-38 levels in plasma can e.g. be determinedby ELISA using a CNP antibody that binds to the ring region of CNP, asdescribed in the literature (U.S. Pat. No. 8,377,884 B2), or byLC-MS/MS.

Results: Dose administrations were well tolerated with no visible signsof discomfort during administration and following administration. Nodose site reactions were observed any time throughout the study. Afterdose administration the total CNP-38 t_(max) was observed at 12 h forcompound 10f and 24 h for compound 11i. Total CNP-38 plasma levels werebelow LOQ (100 ng/mL, C-terminal peptide) after 120 h for compound 10f,while the plasma level was still as high as ca. 30% of c_(max). forcompound 11i after 168 h (C-terminal peptide). For compound 11i similarterminal half life of 3-4 d was found for the C-terminal and theN-terminal peptide, indicating the presence of intact CNP-38 in theconjugate.

Conclusion: The favourable long lasting PK over several days and thestability of CNP-38 in the conjugate 11i indicates its suitability asCNP-38 prodrug for providing long lasting levels of released bioactiveCNP-38 over several days.

Example 27

Synthesis of Fluorescein Labelled CNP-38 27d and NPR-C Affinity Assay

Compound 27d was synthesized according to the following scheme:

Side chain protected CNP-38 on TCP tentagel resin having free N-terminus27a (0.50 g, 35.4 μmol) was pre-swollen in DMF for 30 min. A solution of5(6)-carboxyfluorescein-N-succinimidyl ester (41.9 mg, 88.5 μmol) andDIEA (30.9 μL, 177 μmol) in DMF (1.6 mL) was drawn onto the resin andthe mixture was shaken over night at rt. The resin was washed 10× eachwith DMF and CH₂Cl₂ and dried in vacuo affording 27b.

Cleavage of the peptide from resin and removal of protecting groups wasachieved by treatment of the resin with 7 mL cleavage cocktail100/3/3/2/1 (v/w/v/v/v) TFA/DTT/TES/water/thioanisole for 1 h at rt. Theresin was filtered off and crude 27c was precipitated in pre-cooled(−18° C.) diethyl ether and purified by RP-HPLC affording 27c. Thecombined HPLC fractions were used directly in the next step.

MS: m/z 1105.80=[M+4H]⁴⁺, (calculated monoisotopic mass for[M+4H]⁴+=1105.81).

To the combined HPLC product fractions of 27c (115 mL), 30 mL of 0.5 Mcitric acid buffer (pH=5.00) and 8 mL of a 10 mM2,2′-dithiobis(pyridine-N-oxide) solution in 1/1 (v/v)acetonitrile/water were added. The mixture was stirred for 60 min at rtand then diluted with 350 mL of water containing 0.1% TFA (v/v). 27d waspurified by RP-HPLC.

Yield: 16.1 mg (2.90 μmol, 8.2% over 3 steps) labelled CNP-38*10 TFA

MS: m/z 1105.30=[M+4H]⁴⁺, (calculated monoisotopic mass for[M+4H]⁴⁺=1105.30).

For the NPR-C affinity assay, a NPR-C expressing Hek293 cell line wasdeveloped. The coding region of the NPR-C sequence (BC131540) was clonedinto a lentiviral vector under CMV promoter for constitutive receptorexpression. A bicistronic element located on the vector for puromycinresistence was used as eukaryotic selection marker. After transduction,stably growing cell pools were subjected to qRT-PCR for confirmation ofreceptor mRNA-expression compared to parental Hek293 cells. AnNPR-C-expressing cell pool was expanded and frozen as master cell bankfor CNP sample testing.

For the assay, growing cells were trypsinized from the cell flaskbottom, counted, and seeded in a 96-well plate (1.5×10⁵/well) andcentrifuged. Supernatants were discarded. CNP standard and sample wereserially diluted over 9 steps in PBS 0.2% BSA and transferred to themicro plate in duplicates and mixed with cells. After 30 min incubationat room temperature, fluorescein-labelled CNP 27d was added to each wellwith a constant concentration and cells were incubated for additional 45min at room temperature.

Subsequently, cells were analyzed by flow cytometry using meanfluorescence intensity of the FITC channel (FL1, Beckman CoulterFC500MPL) as read out.

Standard curve and sample curve were generated in an analysis software(PLA 2.0) using a 4 PL fit for potency and/or IC₅₀ calculation.

TABLE 3 Residual NPR-C affinity of PEGylated CNP-38 variants in acell-based assay versus CNP-38 IC₅₀ of PEGylated CNP-38/ CompoundPEGylation IC₅₀ CNP-38 15e 5 kDa PEG, N-Terminus 0.53 19e 5 kDa PEG,Lys26 1.1 10f 2 × 20 kDa PEG, Lys26 12 (reversible conjugate, firstcarrier branching point close to CNP moiety) 12g 4 × 10 kDa PEG, Lys26143 (permanent conjugate, first carrier branching point close to the CNPmoiety) 11i 4 × 10 kDa PEG, Lys26 91 (reversible conjugate, firstcarrier branching point close to the CNP moiety) 31d 4-arm PEG 40 kDa,Lys26 1.7 (reversible conjugate, first carrier branching point not closeto the CNP moiety)

Example 29

Synthesis of Asn-Linker Reagent 29b

Asn-linker reagent 29b was synthesized according to the followingscheme:

To a solution of 1b (12.85 g, 16.14 mmol) in isopropanol (238 mL), H₂O(77.5 mL) and LiOH (2.32 g, 96.87 mmol) were added. The reaction mixturewas stirred for 4 h at rt. Afterwards, the reaction mixture was dilutedwith toluene (300 mL). The phases were separated and the organic phasewas washed 3× with 0.1 M HCl (200 mL). The phases were separated again.The aqueous phase was extracted 3× with toluene (100 mL). The productwas found in the acidic aqueous phase and the pH value of this phase wasadjusted to pH 8.5 by the addition of 4 N NaOH. Then, the aqueous phasewas extracted 3× with CH₂Cl₂ (200 mL). The organic phase was washed withbrine (50 mL), dried over Na₂SO₄ and filtrated. 29a was isolated uponevaporation of the solvent and used in the next reaction without furtherpurification.

Yield: 6.46 g (13.36 mmol, 83%)

MS: m/z 484.06=[M+H]⁺, (calculated monoisotopic mass=483.26).

To a solution of 6-maleimidohexanoic acid (1.73 g, 8.19 mmol) in THF (70mL), PyBOP (4.26 g, 8.19 mmol) and DIEA (3.89 mL, 22.33 mmol) wereadded. Then, the reaction mixture was stirred for 2 h at rt. Afterwards,29a (3.60 g, 7.44 mmol) was dissolved in THF (10 mL) and added to thereaction mixture. The reaction was stirred at rt overnight. Then,methyl-tert-butylether (300 mL) was added. The organic phase was washed2× with 0.1 M HCl solution (200 mL). The combined aqueous phases wereextracted 2× with methyl-tert-butylether (200 mL). The combined organicphases were washed with brine (150 mL), dried over Na₂SO₄ and filtrated.The solvent was evaporated in vacuo. 29b was purified using flashchromatography.

Yield: 3.34 g (4.94 mmol, 66%)

MS: m/z 677.34=[M+H]⁺, (calculated monoisotopic mass=676.33).

Example 30

Synthesis of 4-Arm-Thiol-PEG 40 kDa 30c

To a solution of 6-tritylmercapto-hexanoic acid (111.72 mg, 286.02μmol), HOBt (43.81 mg, 286.06 μmol) and EDC*HCl (54.84 mg, 286.06 μmol)in CH₂Cl₂ (5 mL) was added 2,4,6-collidine (251 μL, 1.91 mmol). Then,this solution was added to a solution of 4-arm amino PEG 40 kDa (NOF,Sunbright PTE-400PA, 1.30 g, 31.78 μmol) in CH₂Cl₂ (10 mL). The reactionmixture was stirred over night at rt. Afterwards, the solvent wasevaporated (water bath 30° C.). 30b was purified by RP-HPLC.

Yield: 650.5 mg (48%).

Cleavage of the Trt protecting group was achieved by adding the cleavagecocktail (DTT 500 mg/TFA 500 μL/water 500 μL, TES 2.5 mL/HFIP 5.0mL/CH₂Cl₂ 25.0 mL) to 30b (500 mg, 11.79 μmol) and incubating for 30 minat rt. 30c was obtained after precipitation in pre-cooled (−18° C.)diethyl ether.

Yield: 401.3 mg (82%; 93.3% purity).

Example 31

Synthesis of Conjugate 31d

Conjugate 31d was synthesized according to the following scheme:

A solution of linker reagent 29b (3.82 g, 5.64 mmol), OxymaPure (802 mg,5.64 mmol) and DIC (868 μL, 5.64 mmol) in DMF (42.5 mL) was added to theresin 10a (18 g, 1.85 mmol).

The suspension was shaken for 100 min at rt to afford resin 31a. Theresin was washed 10× with DMF (10 mL) and 10× with DCM (10 mL) and driedin vacuo for 15 min. Cleavage of the peptide from resin and removal ofprotecting groups was achieved by treatment of the resin with 135 mLcleavage cocktail 100/3/2/1 (v/v/v/v) TFA/TES/water/thioanisol. Themixture was agitated for 60 min at rt. Crude 31b was precipitated inpre-cooled diethyl ether (−18° C.).

The precipitate was dissolved in TFA (423 mL). To this solution, asolution of diphenyl sulfoxide (1.87 g, 9.25 mmol) and anisole (5.05 mL,46.25 mmol) in TFA (40 mL) was added. Afterwards, trichloromethylsilane(13.3 mL, 114.7 mmol) was added and the reaction mixture was stirred for15 min at rt. Then, ammonium fluoride (10.96 g, 296 mmol) was added andthe solution was stirred for 2 min in a water bath at rt. Crude 31c wasprecipitated in pre-cooled diethyl ether (−18° C.) and purified byRP-HPLC.

Yield: 187 mg (34.2 μmol, 16%) CNP-38-linker*9 TFA

MS: m/z 1110.33=[M+4H]⁴⁺, (calculated monoisotopic mass for[M+4H]⁴⁺=1110.33).

To a solution of 31c (88.0 mg, 16.1 μmol) in 4.40 mL MeCN/H₂O (1:1)containing 0.1% TFA (v/v) was added a solution of 4-arm-thiol-PEG 40 kDa30c (107.35 mg, 2.59 μmol) in 1.45 mL water containing 0.1% TFA and 1 mMEDTA, followed by 0.5 M phosphate buffer containing 3 mM EDTA (1.46 mL,pH 6.0). The mixture was incubated for 2 h at rt. Conjugate 31d waspurified by RP-HPLC.

Yield: 129 mg (2.09 μmol, 80%) conjugate 16d*36 TFA

Example 32

Alternative Synthesis of Dmb Protected 6-Mercaptohexanoic Acid 7

Dmb-protected mercapto hexanoic acid 7 was synthesized according to thefollowing scheme:

To a solution of 6-bromohexanoic acid (100 g, 0.513 mol) in EtOH (1.0 L)was added thiourea (47 g, 0.615 mol) in one portion at 20° C. Then, thesuspension was heated up to 78° C. (a clear solution was formed) andstirred for 12 h. A solution of NaOH (62 g, 1.54 mol) in H₂O (1.0 L) wasadded dropwise with a constant pressure funnel. Afterwards, refluxingwas continued for additional 2 h. The reaction mixture was poured intoH₂O (1 L) and extracted with EtOAc (1 L). The aqueous phase wasacidified with con. HCl towards pH=2 and then extracted 3× with EtOAc(500 mL). The combined organic phases were washed with brine (400 mL).Afterwards, the combined organic phases were dried over Na₂SO₄,filtrated and the solvent was evaporated under reduced pressure at 45°C. The 6-mercaptohexanoic acid was used in the next reaction withoutfurther purification.

Yield: 62 g (crude)

¹H-NMR (400 MHz, CDCl₃):

δ=2.50-2.55 (q, J=7.2 Hz, 2H), 2.36 (t, J=7.6 Hz, 2H), 1.66-1.61 (m,4H), 1.41-1.49 (m, 2H), 1.34 (t, J=7.6 Hz, 1H) ppm.

6-mercaptohexanoic acid (27.0 g, 0.182 mol) was charged in a 1 Lthree-necked bottom flask with anhydrous THF (540 mL). The solution wasdegassed by freeze-pump-thaw technique and then cooled to 0° C. with anexternal ice bath. NaH (18.2, 455.4 mmol, 4.16 mL, 60% purity) was addedwith spoon horns over 30 min at 0° C. Then, 2,6-dimethylbenzylchloride(28.2 g, 0.182 mol) was added in one portion. The reaction mixture waswarmed up to 20° C. and stirred for 12 h. The reaction mixture waspoured into H₂O (540 mL) and extracted 2× with MTBE (540 mL).Afterwards, the aqueous phase was acidified with conc. HCl towards pH=2and then extracted 3× with MTBE (500 mL). The combined organic phaseswere washed with brine (500 mL), dried over Na₂SO₄ and filtrated. 7 wasisolated upon evaporation of the solvent under reduced pressure at 45°C. as a yellow oil.

Yield: 41.5 g (0.16 mol, 85%)

¹H-NMR (400 MHz, DMSO-d₆):

δ=11.99 (s, 1H), 7.05-7.07 (d, J=6.8 Hz, 1H), 6.97 (s, 1H), 6.91-6.92(d, J=6.8 Hz, 1H), 3.66 (s, 2H), 2.38-2.39 (m, 2H), 2.29 (s, 3H), 2.23(s, 3H), 2.16-2.19 (m, 2H), 1.40-1.55 (m, 4H), 1.22-1.38 (m, 2H) ppm

MS (neg. mode): m/z 265.0=[M−H]⁻, (calculated monoisotopic mass=265.13).

Example 33

Synthesis of Linker Reagent 33c

Linker reagent 33c was synthesized according to the following scheme:

Four reactions were carried out in parallel. To a solution of compound1b (60 g, 75 mmol) in CH₂Cl₂ (300 mL) was added piperidine (58 g, 0.68mol, 67 mL). The reaction mixture was stirred at rt for 4 h. The fourreactions which were performed in parallel were combined for work-up.The reaction mixture was diluted with H₂O (500 mL) and adjusted with a0.5 N HCl solution towards pH=3˜4. The organic phase was separated andthe aqueous phase was extracted with CH₂Cl₂ (800 mL). The combinedorganic phases were washed with brine (400 mL) and 5% saturated NaHCO₃solution (400 mL) in turn. Then, the combined organic phases were driedover Na₂SO₄, filtered and the solvent was evaporated in vacuo. 33a waspurified by chromatography on silica (100-200 mesh) with DCM/MeOH (20/1to 4/1).

Yield: 150 g (87%)

¹H-NMR (400 MHz, DMSO-d₆):

δ=7.34-7.38 (m, 4H), 6.25-7.29 (m, 2H), 5.08-5.19 (m, 2H), 4.60-4.68 (m,1H), 4.32-4.40 (m, 2H), 3.73-3.79 (m, 9H), 3.10-3.27 (m, 3H), 2.65-3.05(m, 8H), 1.36 (s, 9H) ppm.

Two reactions were carried out in parallel. To a solution ofFmoc-L-Lys(Fmoc)-OH (79 g, 0.13 mol), 33a (70 g, 0.12 mol),4-ethyl-morpholine (70 g, 0.61 mol, 77 mL) in MeCN (850 mL) was addeddropwise T₃P (50% in EtOAc; 140 g, 0.22 mol) over a period of 30 min.After addition, the reaction mixture was stirred at rt for 18 h. The tworeactions which were performed in parallel were combined for work-up.The reaction mixture was diluted with H₂O/CH₂Cl₂ (1:1, 2 L) and thenadjusted with 0.5 N HCl solution towards pH=3-4. The organic phase wasseparated and the aqueous phase was extracted with CH₂Cl₂ (1 L). Thecombined organic phases were washed with brine (800 mL) and 5% NaHCO₃solution (800 mL) in turn. Then, the combined organic phases were driedover Na₂SO₄, filtered and the solvent was evaporated in vacuo. 33b waspurified by chromatography on silica (100-200 mesh) with petroleumether/ethyl acetate (5/1 to 1/1).

Yield: 160 g (57%)

¹H-NMR (400 MHz, DMSO-d₆):

δ=7.80-7.90 (m, 4H), 7.61-7.68 (m, 5H), 7.20-7.40 (m, 14H), 6.14-6.28(m, 3H), 5.01-5.07 (m, 2H), 4.15-4.36 (m, 8H), 3.71-3.77 (m, 9H),2.80-3.53 (m, 9H), 2.66-2.75 (m, 4H), 2.36-2.39 (m, 1H), 1.52-1.55 (m,2H), 0.88-1.19 (m, 13H) ppm.

Two reactions were carried out in parallel. To a solution of 33b (60 g,52 mmol) in MeOH (1.2 L) was added 10% Pd/C (18 g) in a 2 L hydrogenatedbottle. The reaction mixture was degassed and purged 3× with H₂ and thenstirred at 25° C. under H₂-atmosphere (45 psi) for 2.5 h. The tworeactions which were performed in parallel were combined for work-up.The reaction mixture was filtered by diatomite and the filtrate wasconcentrated in vacuo to give crude 33c. 33c was purified bychromatography on silica (100-200 mesh) with DCM/MeOH (200/1 to 100/3).

Yield: 70 g (63%)

¹H-NMR (400 MHz, DMSO-d₆):

δ=12.15 (s, 1H), 7.87-7.89 (m, 4H), 7.50-7.70 (m, 5H), 7.31-7.40 (m,9H), 6.20-6.23 (m, 2H), 4.13-4.36 (m, 10H), 3.70-3.77 (m, 9H), 2.62-3.10(m, 12H), 2.30-2.34 (m, 1H), 2.14-2.18 (m, 1H), 1.50-1.58 (m, 2H),1.25-1.34 (m, 13H) ppm

MS: m/z 1056.4=[M+H]⁺, (calculated monoisotopic mass=1056.50).

Example 34

Alternative Synthesis of 11c

Compound 11c was synthesized according to the following scheme:

A solution of linker reagent 33c (3.21 g, 3.04 mmol), PyBOP (1.58 g,3.04 mmol) and DIPEA (848 mL, 4.86 mmol) in DMF (24.0 mL) was incubatedfor 5 min at rt, then added to the resin 10a (12 g, 1.21 mmol). Thesuspension was shaken for 2.5 h at rt. The resin was washed 10× with DMF(10 mL) and 10× with DCM (10 mL) and dried in vacuo for 60 min.

Example 35

CNP-38 and Conjugate 11i: Evaluation of Cardiovascular Effects in theConscious Mouse (Subcutaneous Administration)

The purpose of this study was to evaluate the haemodynamic side effectsof 11i at dose level equivalent to a CNP-38 dose level elicitinghaemodynamic side effects (decrease in blood pressure) in thetelemetered mouse.

Male Crl:CD1(ICR) mice (age range 8-13 weeks and body weight range27.3-35.6 g at start of dosing) were surgically implanted with aTA11PA-C10 telemetry transmitter (Data Sciences International (DSI)) inthe carotid artery. The body of the transmitter was placedsubcutaneously in the lateral flank of the mouse. The mice were dosedsubcutaneously in a latin square crossover design with at least 72 hoursbetween dosing occasions. Mice were dosed with 1) vehicle (10 mMsuccinate, 46.0 g/L mannitol, pH 4.00), 2) CNP-38 (800 μg CNP-38/kg, 10mM succinate, 46.0 g/L mannitol, pH 4.00) or 3) 11i (800 μg CNP-38eq/kg, 10 mM succinate, 46.0 g/L mannitol, pH 4.00). At least four micewere included at each dose level. Blood pressure (systolic (SAP),diastolic (DAP) and mean (MAP) and heart rate (HR, derived from bloodpressure), were recorded using a digital data capture system linked witha DSITM Ponemah data acquisition and analysis system. The capture systemallowed recording of the cardiovascular parameters whilst the mice werein individual cages. On the day of each test session the animals wereweighed and a predose recording was performed for at least 60 min priorto dosing. Each mouse was returned to the home cage and thecardiovascular parameters were recorded for approximately 48 hourspostdose. Blood pressure and HR were reported at the following timepoints: −30, −20, −10, 5, 15 and 30 min postdose and 1, 2, 6, 12, 18,24, 30, 36, 42 and 48 hours postdose. Each time point was presented asthe average value of five minute's recording prior to the time point.The monitoring period was selected to cover exposure to the test itemsboth prior to and after T_(max).

Results: Compared to predose values, vehicle dosed animals had increasedMAP at the 5, 15, and 30 min post dose sampling time point. This wasconsidered a normal physiological response due to handling and dosing.The same physiological increase in MAP was seen for animals dosed with11i at the 5, 15, and 30 min post dose sampling time point predose. In 3of 4 animals dosed with CNP-38 the physiological increase in MAP was notevident. On the contrary, 3 of 4 CNP-38 dosed animals showed asignificant decrease in MAP at the 5, 15, and 30 min post dose samplingtime point. During the remaining ten time points there were nodifference in MAP between animals dosed with vehicle, CNP-38 and 11i.

MAP (mmHg) predose to 30 min post dose (mean±SD)

Vehicle (n = 10) 11i (n =4 ) CNP-38 (n = 4) predose 101.9 ± 10.0 106.4 ±10.7 106.8 ± 13.4   5 min post dose 125.9 ± 7.3  122.8 ± 5.9  102.0 ±7.5  15 min post dose 126.3 ± 6.9  121.5 ± 7.5  89.5 ± 29.4 30 min postdose 114.4 ± 15.3 111.5 ± 13.7 99.5 ± 25.2

Similar trends were seen for SAP and DAP for all dose levels. HR was notimpacted by treatment with CNP-38 or 11i.

In conclusion, subcutaneous administration of 11i did not decrease bloodpressure as seen for an equivalent dosage CNP-38.

Example 36

Pharmacokinetic Profile of CNP-38 after Subcutaneous Single-DoseAdministration to Cynomolgus Monkeys

This study was performed in order to test the pharmacokinetics of CNP-38after subcutaneous (s.c.) administration in cynomolgus monkeys. Threemale monkeys (2-4 years old, 3-5 kg) received a single s.c. injection ata dose of 40 μg/kg of CNP-38. Blood samples were collected at 5, 10, 15,30, 45 min and 1, 2, 4, 8 hours upon dose.

Method: Plasma levels of CNP were analysed using a commerciallyavailable competitive radioimmuno-assay (RK-012-03, PhoenixPharmaceuticals, CA). The assay was applied essentially as described bythe manufacturer. The assay is based on competitive binding between125I-labelled CNP (supplied in the kit) and unlabeled CNP (from studysample or calibrants) to an anti-CNP antibody. When the concentration ofCNP in the sample increases, the amount of 125I-labelled CNP that isable to bind to the antibody decreases. By measuring the amount of125I-labelled CNP bound as a function of the concentration of peptide,it is possible to construct a calibration curve from which theconcentration of peptide in the sample can be determined.

A few changes to the supplied assay protocol were made. These changesincluded using in-house CNP calibrant and QC samples to secureconsistency between assay runs. In order to shorten the duration of theassay, the initial incubation of samples with antibodies was performedat room temperature for 5 hours (instead of 16-24 hours at 4° C.). Dueto matrix effects in monkey plasma, the minimal required dilution wasset at 1:10, yielding an assay range of 150-1080 pg/mL CNP.

Results: Administration of CNP-38 to cynomolgus monkeys was welltolerated. After s.c. injection, the CNP-38 median T_(max) was observedat 10 min, with a mean half-life time of approximately 7 min.

PK Parameter Result T_(max) (median) 10 min C_(max) (mean) 7.9 ng/mLAUC_(tlast) (mean) 2.5 h*ng/mL Half-life (mean) 6.6 min

Example 37

Pharmacokinetic Profile of Conjugate 11i after Subcutaneous Single-DoseAdministration to Cynomolgus Monkeys

This study was performed in order to investigate pharmacokinetics of 11iafter s.c. administration in cynomolgus monkeys. Four male animals (2-4years old, 3-5 kg) received a single s.c. injection of 11i at a dose of40 μg CNP-38 eq/kg. Blood samples were collected up to 168 h post doseand plasma was generated (LiHeparin). Total CNP-38 concentrations weredetermined by LC-MS/MS

Method: The term “total CNP-38” refers to a combination of both freeCNP-38 and CNP-38 bound in the CNP-38 conjugate. Plasma total CNP-38concentrations were determined by quantification of the C-terminalsignature peptide (sequence: IGSMSGLGC) after tryptic digestion anddisulfide bridge reduction.

LC-MS analysis was carried out by using an Agilent 1290 UPLC coupled toan Agilent 6460 Triple Quad mass spectrometer via an ESI probe.Chromatography was performed on a Waters Acquity BEH C18 analyticalcolumn (50×1.0 mm I.D., 1.7 μm particle size, 130 Å) with pre-filter ata flow rate of 0.5 mL/min (T=45° C.). Water (Ultrapure ≤500 ppt sodiumgrade) containing 0.1% formic acid (v/v) was used as mobile phase A andacetonitrile (ULC/MS grade) with 0.1% formic acid as mobile phase B. Thegradient system comprised a short isocratic step at the initialparameters of 0.1% B for 0.5 min followed by a linear increase from 0.1%B to 30% B in 1.5 min. Mass analysis was performed in the multiplereaction monitoring (MRM) mode, monitoring the reactions of theionsation m/z 824.5 [M+H]¹⁺ to 515.2. As internal standard deuteratedCNP-38 conjugate was used.

Calibration standards of CNP-38 conjugate in blank plasma were preparedas follows: The thawed Li-heparin cynomolgus plasma was firsthomogenized, then centrifuged for 5 minutes.

The CNP-38 conjugate formulation was diluted to eight differentcalibration working solutions containing between 0.103 and 51.28 μg/mL(CNP-38 eq.) in 50% methanol/50% water/0.1% formic acid (v/v/v). Theworking solutions were spiked into blank plasma at concentrationsbetween 10.3 ng/mL (CNP-38 eq.) and 5128 ng/mL (CNP-38 eq.). Thestandards were used for the generation of a calibration curve. Acalibration curve was generated based on analyte to internal standardpeak area ratios using weighted (1/x²) linear regression and the sampleconcentrations were determined by back-calculation against thecalibration curve.

For sample preparation, protein precipitation was carried out byaddition of 200 μL of precooled (0° C.) acetonitrile to 50 μL of theplasma sample and 10 μL of internal standard solution (2.8 μg/mL CNP-38eq. in 50% methanol/50% water/0.1% formic acid (v/v/v)). 200 μL of thesupernatant were transferred into a new well-plate and evaporated todryness (under a gentle nitrogen stream at 35° C.). For reconstitutionsolvent 100 μg Trypsin (order number V5111, Promega GmbH, Mannheim,Germany) were dissolved in 100 μL 10 mM acetic acid. 2.5 mL Tris bufferand 500 μL methanol were added. 50 μL of the resulting reconstitutionsolvent were added to each cavity of the-well plate. After 3 hoursincubation at 37° C. (Eppendorf ThermoMixer with ThermoTop), 5 μL of a0.5 M TCEP solution were added to each cavity and incubated again for 30min at 37° C. After the samples had cooled to room temperature, 2 μL 60%formic acid in water were added. 10 μL were injected into the UHPLC-MSsystem. Results: Administration of 11i to cynomolgus monkeys was welltolerated. After s.c. injection the 11i median T_(max) was 36 h, andwith a mean half-life time of 107 h.

PK Parameter Result T_(max) (median) 36 hours C_(max) (mean) 316 ng/mLAUC_(tlast) (mean) 38,051 h*ng/mL Half-life (mean) 107 hours

Example 38

Functional cGMP Stimulation in NIH-3T3 Cells with Released CNP

11i was incubated under physiological conditions (1 mg CNP-38 eq/mL), asdescribed in Example 20. After 7 d, released CNP-38 was isolated byRP-HPLC and analyzed for bioactivity as described in Example 21.

CNP EC₅₀ compound/ Compound Variant PEGylation EC₅₀ CNP-38 ReleasedCNP-38 CNP-38 — 1

Example 39

Alternative Synthesis of 11h

Alternative synthesis of compound 11h: 39a is synthesized by solid phasesynthesis as described in Material and Methods. Protecting group PG1 forthe ring lysin side chain and protecting groups PG2 for the cysteineside chains is Mmt. Mild resin cleavage and disulfide formation byiodine treatment affords compound 39c. After coupling of linker molecule39d and global deprotection, 11h is purified by RP-HPLC.

Abbreviations:

-   ACH achondroplasia-   ACN acetonitrile-   AcOH acetic acid-   AUC_(tlast) Area Under the Curve to the last quantifiable time point-   Bn benzyl-   Boc tert-butyloxycarbonyl-   BSA bovine serum albumin-   cGMP cyclic guanosine monophosphate-   C_(max) Maximum concentration-   CMV cytomegalovirus-   CNP C-type natriuretic peptide-   COMU    (1-cyano-2-ethoxy-2-oxoethylidenaminooxy)dimethylamino-morpholino-carbenium    hexafluorophosphate-   conc. Concentrated-   d day-   CTC Chlorotritylchloride polystyrol-   DAP Diastolic arterial pressure-   DBU 1,3-diazabicyclo[5.4.0]undecene-   DCC N,N′-dicyclohexylcarbodiimide-   DCM dichloromethane-   DIC N,N′-diisopropylcarbodiimide-   DIEA N,N-diisopropylethylamine-   DIPEA N,N-diisopropylethylamine-   DMAP dimethylamino-pyridine-   DMEM Dulbecco's modified Eagle's medium-   Dmb 2,4-dimethylbenzyl-   DMEM Dulbecco's modified eagle medium-   DMF N,N-dimethylformamide-   DMSO dimethylsulfoxide-   DTT dithiothreitol-   EC50 half maximal effective concentration-   EDC 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide-   EDTA ethylenediaminetetraacetic acid-   ELISA enzyme-linked immunosorbent assay-   eq stoichiometric equivalent-   ESI-MS electrospray ionization mass spectrometry-   Et ethyl-   EtOAc ethyl acetate-   EtOH ethanol-   FBS fetal bovine serum-   FGFR3 fibroblast-growth-factor-receptor 3-   FITC fluorescein isothiocyanate-   Fmoc 9-fluorenylmethyloxycarbonyl-   h hour-   HATU O-(7-azabenzotriazole- 1-yl)-N,N,N′,N′-tetramethyluronium    hexafluorophosphate-   HCH hypochondroplasia-   HFIP hexafluoroisopropanol-   HPLC high performance liquid chromatography-   HOBt N-hydroxybenzotriazole-   HR Heart rate-   IBMX 3-isobutyl- 1-methylxanthine-   iPrOH 2-propanol-   iv intravenous-   ivDde 4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)-3-methylbutyl-   LC liquid chromatography-   LTQ linear trap quadrupole-   Mal 3-maleimido propyl-   MAP Mean arterial pressure-   Me methyl-   MeOH methanol-   min minutes-   Mmt monomethoxytrityl-   MS mass spectrum/mass spectrometry-   MSA methanesulfonic acid-   MTBE methyl-tert-butylether-   Mtt methyltrityl-   MW molecular weight-   m/z mass-to-charge ratio-   NEP neutral endopeptidase-   NHS N-hydroxy succinimide-   NPR natriuretic peptide receptor-   OtBu tert-butyloxy-   PBS phosphate buffered saline-   PEG poly(ethylene glycol)-   PFP pentafluorophenol-   pH potentia Hydrogenii-   Pr propyl-   PyBOP benzotriazol-1-yl-oxytripyrrolidinophosphonium    hexafluorophosphate-   Q-TOF quadrupole time-of-flight-   qRT-PCR quantitative real-time polymerase chain reaction-   RP-HPLC reversed-phase high performance liquid chromatography-   rpm rounds per minute-   rt room temperature-   SIM single ion monitoring-   SAP Systolic arterial pressure-   SEC size exclusion chromatography-   sc subcutaneous-   Su succinimidyl-   T₃P 2,4,6-tripropyl-    1,3,5,2,4,6-trioxatriphosphorinane-2,4,6-trioxide-   TCEP tris(2-carboxyethyl)phosphine-   TCP tritylchloride polystyro-   TD thanatophoric dysplasia-   TES triethylsilane-   TFA trifluoroacetic acid-   THF tetrahydrofuran-   TIPS triisoproylsilane-   T_(max) Time of maximum concentration-   TMEDA N,N,N′N′-tetramethylethylene diamine-   Tmob 2,4,6-trimethoxybenzyl-   TR-FRET time-resolved fluorescence energy transfer-   Trt triphenylmethyl, trityl-   UPLC ultra performance liquid chromatography-   UV ultraviolet-   vs. versus-   ZQ single quadrupole

The invention claimed is:
 1. A controlled-release CNP agonist comprisinga CNP moiety comprising a ring moiety (−D), wherein the ring moiety hasthe amino acid sequence of SEQ ID NO:96, the ring moiety being betweentwo cysteine residues forming a disulfide bridge, and being conjugatedto a polymer via a linker cleavable in aqueous buffer at pH 7.4 and 37°C. in the absence of enzymes with a half-life of up to six months,wherein the polymer is water-soluble and conjugated via the linker to anamino group side chain of a lysine of SEQ ID NO:96 or the polymer iswater-insoluble, wherein D-H is released from the controlled-release CNPagonist by cleavage of the linker with a release half-life of at least 6hours in aqueous buffer at pH 7.4 and 37° C. in the absence of enzymesand which controlled-release CNP agonist has an EC₅₀ that is at least20-fold higher than the EC₅₀ of the D-H.
 2. The controlled-release CNPagonist of claim 1, wherein the controlled-release CNP agonist has anEC₅₀ that is at least 50-fold higher than the EC₅₀ D-H.
 3. Thecontrolled-release CNP agonist of claim 1, wherein thecontrolled-release CNP agonist has an EC₅₀ that is at least 100-foldhigher than the EC₅₀ D-H.
 4. The controlled-release CNP agonist of claim1, wherein the release half-life is at least 24 hours.
 5. Thecontrolled-release CNP agonist of claim 1, wherein the release half-lifeis at least 168 hours.
 6. A pharmaceutical composition comprising thecontrolled-release CNP agonist of claim 1 and at least one excipient.